sonication + capping/dispersion in appropriate medium

Can the medium be ddH2O? @sanhita ray

Dear Dr. Mohammed'

depends on the process of collection and washing, if you centrifuge it the agglomeration occurs. you can separate it using another method.

Yours'

Ahmed

Dear @Ahmed said mahmoud

Thanks for the support, yes i have used centrifugation and washing with double distilled water.

Is that the cause of agglomeration?

If so what are the other methods to adapt in order to prevent the agglomeration, please suggest.

Thanks

Momina

Dear Monia,

Try to wash it more with DW+ ethanol, slow down centrifuge speed and filtrate it using filter paper

Dear Dr. Ahmed Said Mahmoud

At low centrifuge speed, the pellet formation is very less. The washing is done two times with DW and the last wash with ethanol. I would implement the filtration step at the end as you suggest. is Whatman filter paper feasible to serve the purpose?

Regards

Momina

Dear Dr. Monia,

Whatman filter paper No. 2 or 42, and use ethanol after washing to prevent oxides.

Also, you can also use glassrods to physically separate the agglomeration but I don't like this technique it may affect on the nanostructure specially when you have huge amount of carbons from the green synthesis

Regard

Ahmed

Momina Shanwaz Mohammad

Your problem resides in the washing stages with DI or distilled water and perhaps in the sample preparation stage for SEM. You have washed out all the protective ions, stabilizers, surfactants etc and destroyed the stability of the material causing irreversible aggregation and agglomeration. Any washing should be in the pure, particle-free, mother liquor (i.e. the original ions and additives in the system are retained). The same applies for 'characterization' measurements such as dilution for DLS or zeta potential. One question: why wash the system at all?

See the webinar (registration required) for further information:

Dispersion and nanotechnology

https://www.malvernpanalytical.com/en/learn/events-and-training/webinars/W190528DispNanoMat

Also see:

Silver colloids and invisible ink

https://www.malvernpanalytical.com/en/learn/events-and-training/webinars/W150902SilverColloidInvisibleInk.html

Momina Shanwaz Mohammad

2 quotes from those greater than I...:

• 'I think dry nanotechnology is probably a dead-end' Rudy Rucker Transhumanity Magazine (August 2002)
• ‘If the particles are agglomerated and sub-micron it may be impossible to adequately disperse the particles……‘The energy barrier to redispersion is greater if the particles have been dried. Therefore the primary particles must remain dispersed in water….’ J H Adair, E. Suvaci, J Sindel, “Surface and Colloid Chemistry” Encyclopedia of materials: Science and Technology pp 8996 - 9006 Elsevier Science Ltd. 2001 ISBN 0-08-0431526

Never rely on SEM to indicate likely levels of agglomeration.

If it shows no agglomeration, you can be confident that there isn't any. But if it does show agglomeration, that could easily be due to the way the SEM sample was prepared.

thanks John Francis Miller for that input I am in doubt if it has formed nanocomposite material due to the excess carbon material of the plant. how can I know more about nanocomposites? any help here is most appreciable.

This scientific article may be of some assistance in providing more detailed information about the behavior of nanoparticles. At first glance, your scientific experimental results look something like 'cancer research results,' by which I mean that this is an excellent ResearchGate discussion thread question because you seem to be working to resolve a problem of the sort that scientific researchers in the medical sciences are still trying to solve.

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Article Agglomeration of titanium dioxide nanoparticles increases to...

Research Open Access Published: 26 February 2020 Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo Sivakumar Murugadoss, Frederic Brassinne, Noham Sebaihi, Jasmine Petry, Stevan M. Cokic, Kirsten L. Van Landuyt, Lode Godderis, Jan Mast, Dominique Lison, Peter H. Hoet & Sybille van den Brule Particle and Fibre Toxicology volume 17, Article number: 10 (2020) Cite this article4461 Accesses 2 Altmetric Metricsdetails Abstract Background The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo.Results We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1β increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2.In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates.Conclusion Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs. Background Manufactured nanomaterials (NMs) exist as unbound (single) particles, agglomerates, aggregates or as a mixture thereof [1,2,3,4]. This is clearly recognised in the definition of NMs recommended by the European Union (EU) stating “manufactured material containing particles, in anunbound state or as an aggregate or as an agglomerateand where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range1 nm-100 nm” [5]. This definition was proposed for legislative and regulatory purposes with no direct regard to hazard. Although agglomerates and aggregates (AA) are often erroneously considered similar and interchangeably used, they are, however, two different secondary structures of particulate materials. In agglomerates, the particles bind together by weak forces, which are reversible, while, in aggregates, particles fuse irreversibly together [6]. The terms AA attracted in recent years attention among the NM producers, consumers, regulatory authorities and policy makers in view of their potential influence on human health effects [5, 7, 8]. However, no sound scientific data justify that AA may or may not be relevant from a toxicological perspective. This knowledge gap is not only affecting the risk assessment process but also hindering the development of guidelines to regulate NMs in commercial products.Agglomeration in particular, is a ubiquitous phenomenon and its dynamic behaviour poses a great challenge in assessing health impacts [9, 10]. Unlike aggregates, agglomerates are very sensitive to changes in the environment such as pH, ionic strength, presence of proteins and motion of the carrier medium, and can de-agglomerate/agglomerate further depending on the environment [10, 11]. While this induces complex behaviour of NMs in exposure scenarios and in tissue uptake and bio-distribution, influence on toxicity/biological responses remain poorly understood [9, 10].Titanium dioxide (TiO2) is one of the most abundantly produced NMs and is used in food, paints and in personal care products [12, 13]. Humans are increasingly exposed to TiO2 via inhalation, dermal or oral exposure. Based on animal studies, the International Agency for Research on Cancer (IARC) classified TiO2 as a group 2B carcinogen (possibly carcinogenic to humans) [14]. Very recently, the French agency for food, environmental and occupational health and safety (ANSES) banned the use of TiO2 as a food additive (E171) due to its genotoxic potential [15]. While several studies showed that TiO2 NPs can induce adverse effects including DNA damage and chromosomal damage, findings are contradictory [16, 17]. TiO2 NPs are well known for their agglomeration and, so far, extensive efforts have been dedicated at minimizing agglomeration using different dispersion protocols to assess their toxicity despite a lack of evidence that agglomeration influences their toxicity/biological responses.In this study, we aimed to determine the influence of agglomeration state of TiO2 NPs on toxicity/biological effects. Toxicological studies generally suggest that the smaller the size of the primary NPs the greater the toxicity/biological responses [18,19,20,21]. Therefore,we hypothesized that smaller agglomerates of NPs induce stronger toxicity/biological responses compared to their largely agglomerated counterparts. To test this hypothesis, we selected two TiO2 NPs of identical phase, coating and chemical composition but with different primary particle size and compared their toxicity in different agglomeration states using in vitro and in vivo models. Results Dispersions and size characterization of TiO2 NP agglomerate suspensions Our strategy to prepare ad-hoc stable suspensions of TiO2 NPs with different agglomeration states, in the same dispersion medium, was based on the method developed by Guiot and Spalla [22] (illustrated in Additional file 1: Figure S2). Figure 1 shows representative Transmission Electron Microsopy (TEM) micrographs of the freshly prepared TiO2 stock suspensions. The 17 nm sized TiO2 at pH 2 was relatively well dispersed and predominantly existed as small aggregates (indicated as 17 nm-SA) compared to the suspension prepared at pH 7.5, in which particles tend to agglomerate strongly (17 nm-LA). In contrast, 117 nm TiO2 were found to be less agglomerated when dispersed at pH 7.5 (117 nm-SA) and existed as large agglomerates when dispersed at pH 2 solution (117 nm-LA). After dispersion in the respective pH conditions, TiO2 suspensions were sonicated at constant energy (7056 J) and stabilized immediately using bovine serum albumin (BSA, 0.25%). The suspensions dispersed at pH 2 were readjusted to pH 7–7.5 before size characterization and cell/animal exposure.Fig. 1📷Representative TEM micrographs of freshly prepared TiO2 stock suspensions of small (SA) and large agglomerates (LA). 17 nm-SA (a), 17 nm-LA (b), 117 nm-SA (c) and 117 nm-LA (d)Full size imageSizes of TiO2 suspensions are presented in Table 1. TEM analyses showed that the median Equivalent Circle Diameter (ECD) of 17 nm-SA and 117 nm-SA were 18 and 122 nm, respectively. The TiO2 NPs were, thus, in their most dispersed state in these suspensions. Median ECD of the large agglomerates, 17 nm-LA and 117 nm-LA, were 127 and 352 nm respectively, clearly indicating that NPs were more agglomerated in these suspensions. Mean ECD were substantially different: 100 nm for 17 nm-SA; 200 nm for 117 nm-SA; 250 nm for 17 nm-LA; and 500 nm for 117 nm-LA, confirming the overestimation of sizes when means are used. TEM was also applied to measure mean Feret minimum (Feret min) and the measured sizes were slightly different compared to median ECD (Table 1). The mean hydrodynamic diameter (Z-average) measured by dynamic light scattering (DLS) showed larger sizes for 17 nm TiO2 (SA 600 nm; LA 900 nm) than 117 nm stock suspensions (SA 280 nm; LA 580 nm). Hydrodynamic sizes measured using particle tracking analysis (PTA) were smaller than Z-average in sizes for 17 nm TiO2 suspensions (SA 134 and LA 207 nm) and 117 nm TiO2 suspensions (SA 259 and LA 221 nm).Table 1 Size characterization of freshly prepared TiO2 stock suspensions (2.56 mg/mL)Full size tableThe stability of these suspensions in exposure media was measured by DLS (Table 2). After dilution to 100 μg/mL, Z-averages were measured directly and after 24 h. In DMEM/F12 (typically used for HBE cell cultures) and RPMI 1640 (used for THP-1) only a slight change was observed after 24 h of incubation. In DMEM/HG (used for Caco2), at least a two-fold increase of Z-average after 24 h incubation was noted. The polydispersity index (PDI) was less than ~ 0.35 in stock suspensions and in cell culture medium at 0 and 24 h, indicating an acceptable distribution of sizes and good stability of these suspensions.Table 2 Size characterization of TiO2 in stock and exposure media (HBE,Caco2 and THP-1) using DLSFull size tableIn conclusion, TEM indicated a clear difference between SA and LA for both TiO2 NPs and stock suspensions were found to be stable over 24 h using DLS, indicating that these ad-hoc suspensions were appropriate to test our hypothesis.Influence of TiO2 agglomeration on in vitro dosimetry Before examining biological responses to these differently agglomerated suspensions, we considered the possible influence of differential sedimentation of the suspensions in vitro, which might confound the cell responses. In vitro dosimetry simulation was performed only using DMEM/F12 (used for HBE) and RPMI 1640 (used for THP-1) because DMEM/HG (used for Caco2) promoted further agglomeration over 24 h incubation. The main parameters used to perform dosimetry simulation are listed in Additional file 1: Table S1. Figure 2 shows the estimated TiO2 dose reaching the bottom of the wells as a function of nominal (applied) dose. Regardless of the type of exposure medium and TiO2 primary size/agglomeration state, nearly 56–58% of the applied doses was delivered to the bottom of the wells after 24 h. Thus, the delivered doses between SA and their LA suspensions of both TiO2 did not differ substantially. The results are, therefore, presented as a function of nominal doses (expressed in μg/mL).Fig. 2📷Estimated TiO2 dose reaching the bottom of the wells after 24 h as a function of increasing nominal doses applied in exposure media. Dosimetry simulation was performed with a distorted grid (DG) model for 17 (a and c) and 117 nm (b and d) using parameters obtained from exposure media DMEM/F12 (a and b) and RPMI 1640 (c and d). The slope values are indicated near the respective lines. R2 > 0.99 for all the suspensions. The percentage of dose delivered to the cells did not differ for 96 and 24 well plates, as the height of the liquid column was similar (6 mm)Full size imageComparison of biological responses Since inhalation and ingestion are the primary routes of exposure to these NPs during production and use, we studied the in vitro effects in human bronchial (HBE) and colon (Caco2) epithelial cell lines, respectively. In addition, we used a human monocytic cell line (THP-1) as a representative of innate immune cells that are actively involved in phagocytosis of these particles. To investigate the acute toxicity in vivo, oropharyngeal and gavage administrations were used as representative for inhalation and ingestion, respectively.In order to investigate the validity of our hypothesis, we first determined the endpoints for which responses to TiO2 exposure (for both SA and LA suspensions) were statistically different compared to untreated control using one-way ANOVA. Table 3A and B summarise these results in vitro and in vivo, respectively. If no impact of TiO2 treatment on a given endpoint in both agglomeration states (SA and LA) was revealed, such endpoint was not used to test the hypothesis of the influence of agglomerations. In a second step, we analysed only those endpoints where TiO2 induced a significant effect at least in one of the agglomeration states (SA or LA). We compared the effects induced by SA and LA using two-way ANOVA. If differences were observed between suspensions, a post hoc test (Bonferroni’s multiple comparison test) was used to determine the suspension that induces the strongest effect at the same mass concentration/dose (see Table 4A and B).Table 3 Summary of the in vitro (A) and in vivo (B) responses to TiO2 exposureFull size tableTable 4 Summary of in vitro (A) and in vivo (B) responses to differently agglomerated TiO2 suspensionsFull size tableThe results indicated in green (SA = LA) are shown in Additional file 1: Figures S3 to S8. Significant results indicated in red or blue in Table 4, are presented in Fig. 3 for the in vitro experiments (total glutathione, IL-8, IL-1β and DNA damage in THP-1 exposed to 17 nm TiO2) and in Fig. 4 for the in vivo experiments (lymphocytes in the broncho-alveolar lavage and Ti persistence in lung tissue for aspirated mice with 117 nm TiO2, Fig. 4a and b; blood DNA damage in gavaged mice for 17 and 117 nm TiO2, Fig. 4c and d).Fig. 3📷Influence of TiO2 agglomeration on THP-1 biological responses. Total glutathione (GSH) (a), IL-8 (b) and IL-1β secretion (c), and DNA damage (d) measured in cell cultures after 24 h exposure to different concentrations of small (SA) and large agglomerates (LA) of 17 nm TiO2. Data are expressed as means ± SD from three independent experiments performed in duplicates. p 

This is a long scientific research article which I am dividing into 2 parts:

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J Nanobiotechnology. 2017; 15: 48.

Published online 2017 Jun 26. doi: 10.1186/s12951-017-0281-6

PMCID: PMC5485545

PMID: 28651541

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation

Blanka Halamoda-Kenzaoui, Mara Ceridono, Patricia Urbán, Alessia Bogni, Jessica Ponti, Sabrina Gioria, and Agnieszka Kinsner-Ovaskainen📷

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This article has been cited by other articles in PMC.

Data Availability StatementAssociated Data

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Abstract

Background

Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called “critical quality attributes”, that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization.

Results

We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted.

Conclusions

We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12951-017-0281-6) contains supplementary material, which is available to authorized users.

Keywords: Silica nanoparticles, Cell uptake, Endocytosis route, Agglomeration/aggregation, In vitro

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Background

A detailed understanding of the mechanisms of interaction between engineered nanoparticles (NPs) and biological systems is essential to properly assess the safety of newly developed nanotechnological and nanomedicinal products. Upon exposure, NPs may interact with the outer surface of the cellular membrane and subsequently enter the cells by different endocytic routes. Elucidation of the mechanism by which NPs are internalized into the cells can provide insights about the intracellular trafficking, fate and cytotoxic profile of NPs [1]. Targeting of specific cellular structures, the release of NPs by the cells or, on the contrary, their degradation in lysosomes are all key features that can significantly affect the NPs toxicity/safety, but also the efficacy of novel nanomedicines.

All these processes are highly dependent on the mechanism of NP internalisation [2, 3]. The involvement of various endocytic pathways in NP uptake has been investigated in several in vitro studies, and the obtained results show wide variability with respect to cell lines and nanoparticles used [4–6].

It is well recognised that an accurate characterisation of NPs in the biological medium and under the conditions of an in vitro study is necessary to properly interpret the results and to enable correlation of the physicochemical parameters with the biological response [7]. Indeed, NP properties such as size, shape, surface chemistry and charge were shown to affect the level of cellular uptake and the mechanism of the endocytosis [8–11]. However up to date, little attention has been paid to the question of how this process is influenced by the agglomeration state of NPs. Though, the ability to agglomerate is one of the predominant features of a NP suspension. Changes to the pH and ionic strength or the presence of biomolecules, particularly proteins, can easily modify the NP surface properties, leading to the loss of colloidal stability and formation of agglomerates. That is why this study focused on understanding how the agglomeration state of NPs can influence the endocytic mechanism by which NPs can enter the cells. We have observed in our previous study [7] that silicon dioxide NPs showed a time-dependent tendency to agglomerate in the complete medium of CaCo-2 cells. Therefore, in this study we used the same amorphous fluorescent Rubipy-SiO2 NPs of two different sizes, 30 and 80 nm that were either freshly dispersed in complete medium and added to the cells, or pre-incubated with the complete medium for 24 h in order to allow the agglomeration. Subsequently, for the agglomerated and non-agglomerated NPs of both sizes we compared the level of cellular uptake and the endocytosis route in Caco-2 cells. We used flow cytometry to quantify the cellular uptake, transmission electron microscopy (TEM) to visualise the NPs inside the cells and a high-throughput fluorescence microscopy technique to colocalise NPs with clathrin, caveolin-1 and SNX5 antibodies. We also performed blocking of different endocytic routes by chemical inhibitors and by gene silencing to assess the effect on NP internalisation.

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Methods

Nanoparticle synthesis and characterization

The mono-dispersed fluorescent particles of silicon dioxide 30 and 80 nm labelled with Rubipy were synthetized as described previously [7]. Physicochemical characterization of the NPs including size distribution, zeta potential and fluorescence spectrum carried out in water, PBS and cell culture medium was already described [4]. Additionally, the size distribution of Rubipy-SiO2 NPs was determined in complete CaCo-2 cell culture medium by centrifugal liquid sedimentation (CLS) in a sucrose density gradient using a CLS Disc Centrifuge model DC24000UHR (CPS Instruments Europe, Netherlands). The samples were prepared as a 1 mg/ml suspension of Rubipy-SiO2 NPs in cell culture media (CCM) containing 10% of foetal bovine serum, and the measurements were performed either immediately or after 24 h incubation at 37 °C. In addition, the particle size in complete CaCo-2 cell culture medium was evaluated by TEM. Ultrathin Formvar-coated 200-mesh copper grids (Tedpella Inc.) were first functionalized by placing the carbon-coated side on a drop of 20 μl of Alcian blue (2% in water) deposited on a Parafilm. After 10 min incubation the grid was washed 5 times by the deposition on the drops of water placed on a Parafilm and the excess fluid was removed by blotting its edge on a strip of paper tissue, leaving a rest of humidity. Finally the grid was placed on a 20 μl drop of the corresponding sample, incubated for 10 min and the excess of fluid was removed again with a paper tissue. TEM (JEOL JEM 2100, Japan) at an accelerating voltage of 200 kV was used to visualize the nanoparticles.

Before the biological experiments, Rubipy-SiO2 NPs were purified in centrifugal filters (Amicon Ultra, 10K, Milipore, Italy) at 3000 rpm for 10 min at room temperature (RT) and re-suspended in sterile PBS. The fluorescence spectrum of Rubipy-SiO2 NPs before and after the centrifugation was compared and used for the calculation of the NP concentration. The fluorescence intensities of different concentrations of Rubipy-SiO2 NPs in PBS and in complete CaCo-2 medium without phenol red were obtained using fluorescence spectrophotometer (Cary Eclipse, Varian, Australia Pty Ltd). The scan of the fluorescence emission between 500 and 700 nm was carried out at excitation 460 nm.

Cell lines and culture conditions

Colorectal adenocarcinoma CaCo-2 cells (ACC169, DSMZ, Braunschweig, Germany) were cultured under standard cell culture conditions in Dulbecco’s modified Eagle’s medium with high glucose (4.5 g/L), supplemented with 10% heat inactivated Foetal Bovine Serum (North American origin), 1% penicillin–streptomycin, 4 mM l-glutamine and 1% non-essential amino acids. All cell culture reagents were purchased from Thermo Fisher Scientific, Italy. The experiments were performed between passages 1–10 after defrosting of cells from liquid nitrogen storage.

Internalisation of endocytic markers

Cells were seeded in a 24-well plate (Costar, Italy) at a density of 6 × 104 cells/well, grown until 80–90% confluence, washed, and after serum starvation for 2 h, pre-incubated for 30 min with endocytosis inhibitors: chlorpromazine 50 µM, dynasore 80 µM, methyl-beta-cyclodextrin 5 mM, nystatin 40 µg/ml, genistein 200 µM, or EIPA 75 µM (all from Sigma-Aldrich, Italy). The following endocytic markers were then added to the cells for 30 min: Transferrin Alexa Fluor 488 conjugate 50 µg/ml (clathrin-mediated endocytosis), Bodipy-FL C5-Lactosylceramide (LaCer) complexed to bovine serum albumin (BSA) 1 µg/ml (caveolae-mediated endocytosis), and Alexa Fluor 488-labeled Dextran 10 kDa, 200 µg/ml (macropinocytosis) (all from Thermo Fisher Scientific, Italy).

After exposure the cells were washed twice in cold washing buffer (Hepes 10 mM, NaN3 5 mM) and the remaining membrane bound label was removed according to previously published methods [12, 13]. Briefly, 1 min incubation in ice-cold acidic wash (0.2 M acetic acid, 0.2 M NaCl, pH 2.0), followed by two washes in cold washing buffer was employed to remove surface-bound Transferrin. For the LaCer uptake the “back exchange” procedure was applied, consisting on 1 h washing with 5% fatty acid-free BSA in washing buffer at 4 °C changing for fresh BSA every 10 min. After the washing procedure, the cells were detached by trypsinisation and analysed immediately by flow cytometry.

Analysis of uptake of Rubipy-SiO2 NPs by flow cytometry

Cells were seeded in a 24-well plate (Costar) at a density of 6 × 104 cells/well, grown until 80–90% confluence and exposed to 200 µg/ml of 30 and 80 nm Rubipy-SiO2 NPs freshly dispersed in complete medium or pre-incubated in complete medium for 24 h at 37 °C. Depending on the purpose of the assay the exposure was done at 37 °C or at 4 °C. For the study of endocytic pathways before the exposure to Rubipy-SiO2 NPs, the cells were pre-incubated for 30 min with endocytosis inhibitors, as described above. Following the exposure to Rubipy-SiO2 NPs the cells were washed 3 times in PBS, trypsinised and blocked with a complete cell culture medium, washed again in PBS and analysed immediately by flow cytometry.

Evaluation of cell associated fluorescence, forward scattering (FSC) and side scattering (SSC) were carried out using CyFlow space flow cytometer (Partec, Munster, Germany) and the data were analysed using FCS Express 4 software (De Novo, Los Angeles, CA). Laser excitation was 488 nm and emission bandpass wavelength was 590/50 nm for Rubipy-SiO2 NPs related fluorescence. A minimum of 15,000 cells per sample were analysed; cells debris, nanoparticles and doublets were excluded from the analysis by gating on the FSC versus SSC log graph and on the FL-2 area versus FL-2 width graph, respectively. The median cell associated fluorescence after the subtraction of cell autofluorescence was averaged between three independent experiments (2 replicas each). The results were then normalised according to the reference values of fluorescence intensity of 30 and 80 nm Rubipy-SiO2 NPs to allow the comparison of cell uptake of both sizes of NPs. The efficiency of inhibitors is calculated as a percentage of the uptake by the control cells without any inhibitor.

Analysis of uptake of Rubipy-SiO2 NPs by transmission electron microscopy

Following 3 and 6 h of exposure to 200 µg/ml of 30 and 80 nm Rubipy-SiO2 NPs freshly dispersed in complete medium or pre-incubated in complete medium for 24 h at 37 °C, the cells were washed 3 times in PBS, trypsinised, blocked with a complete cell culture medium and washed again 3 times in PBS by centrifugation (200×g, 5 min). The supernatant was discarded and the cells were fixed using a Karnovsky 2% v/v solution (glutaraldehyde and paraformaldehyde in 0.05 M cacodylate, pH 7.3, Sigma-Aldrich, Italy) over night. Cells were then washed 3 times with 0.05 M cacodylate, pH 7.3 and post-fixed in osmium tetroxide solution in 0.1 M cacodylate, pH 7.3 (both from Sigma-Aldrich, Italy) for 1 h. After 3 washes in cacodylate 0.05 M of 10 min each, cells were dehydrated in a graded series of ethanol solution in MilliQ water (30; 50; 75; 95% for 15 min each, and 100% for 30 min), incubated in absolute propylene oxide (Sigma-Aldrich, Italy) for 20 min (2 changes of 10 min each) and embedded in a solution of 1:1 epoxy resin (Sigma-Aldrich, Italy) and propylene oxide for 90 min. This mixture was renewed with pure epoxy resin (Sigma-Aldrich, Italy) over night at room temperature and later polymerized at 60 °C for 48 h. Ultrathin sections (60–90 nm) were obtained using Leica UCT ultramicrotome (Leica, Italy) and stained with uranyl acetate for 25 min and lead citrate for 20 min (both from Sigma-Aldrich, Italy), washed and dried. Ultrathin sections were imaged using JEOL-JEM 2100 TEM (JEOL, Milan, Italy) at 120 kV.

Colocalisation with endocytic proteins

Cells were seeded in a 96-well plate (black, glass bottom, Greiner Bio-one) at a density of 1 × 104 cells/well and grown until 80–90% confluence was reached. Afterwards the cells were exposed to 200 µg/ml of 30 or 80 nm Rubipy-SiO2 NPs freshly dispersed in complete medium or pre-incubated in complete medium for 24 h at 37 °C. After 3 h exposure the cells were washed 3 times in PBS, fixed with 3.7% paraformaldehyde in PBS, permeabilised with cold methanol at −20 °C and blocked for 30 min at RT with 3% BSA in PBS. Overnight incubation at 4 °C with primary antibodies: either anti-clathrin heavy chain (Abcam, rabbit polyclonal, 1:2000), anti-caveolin-1 (Abcam, rabbit polyclonal, 1:800) or anti-SNX5 (Abcam, goat polyclonal, 1:90) was followed by 45 min incubation at 37 °C with secondary antibodies: anti-rabbit IgG (Invitrogen, goat, Cy® 5 conjugate, 1:300), or anti-goat IgG (Invitrogen, rabbit, Alexa Fluor® 647, 1:300) and DAPI (Molecular Probes, 2,5 µg/ml final concentration). Cells were imaged using IN Cell Analyzer 2200 (GE Healthcare). During acquisition, a minimum of 20 fields per well were imaged using a 60× objective. The representative images were selected, DAPI, Cy3 and Cy5 channels were merged and the qualitative analysis of colocalisation between images from channel Cy3 (Rubipy-SiO2 NPs) and from channel Cy5 (secondary antibodies) was performed using ImageJ software.

Transfection of cells with si-RNA

The cells were seeded in a 24-well plate (Costar) at a density of 5 × 104 cells/well and cultured for 24 h to be 30–40% confluent on the day of transfection. The si-RNA procedure was performed according to the manufacturer's protocol using Silencer® Select validated si-RNA and Lipofectamine™ RNAiMAX at the concentrations selected from the optimization assay: 5 nM si-RNA (CAV1 silencing, Ambion, s2446), or 8 nM si-RNA (PAK1 silencing, Ambion, s10019) and 1 µl Lipofectamine/well. In parallel, a negative control si-RNA was used at the same concentration. Both si-RNA and Lipofectamine were diluted in Opti-MEM® I Reduced Serum Medium, combined, mixed gently and incubated for 15 min at RT before being added drop-wise to the wells. All reagents were purchased from Thermo Fisher Scientific, Italy. After 24 h incubation the medium in the wells was exchanged with the standard cell culture medium containing 10% of serum and no antibiotics. 48 h after transfection the silencing efficiency was tested using TaqMan® gene expression assay and the protein expression was evaluated by western blot method. The cell uptake of Rubipy-SiO2 NPs was quantified by flow cytometry after 3 and 6 h exposure in cells transfected with silencing-RNA in comparison to the cells transfected with negative control si-RNA.

Real-time PCR amplification

Following transfection cells were washed with cold PBS and lysed in 350 μl RLT lysis buffer (Qiagen, Germantown, MD, USA). Samples were stored at −80 °C until RNA extraction was carried out. RNA was extracted from cells and purified using the RNeasy Plus kit (Qiagen, Germantown, MD, USA). The RNA quantification was done by a ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies), and the RNA integrity was assessed with the Agilent 2100 Bioanalyzer (Agilent), according to the manufacturer’s instructions. All RNA samples used in this study had a 260/280 ratio above 1.9 and a RNA Integrity Number (RIN) above 9.0.

1 µg of total RNA was reverse transcribed using the high-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Italy), following the manufacturer's protocol. Real-Time PCR was performed with a total of 10 ng of cDNA for each reaction, using TaqMan® gene expression assay for CAV1 (Hs00971716_m1, Applied Biosystems), for PAK1 (Hs00945621_m1, Applied Biosystems) for GAPDH as a negative control (Hs0275899_g1, Applied Biosystems) and TaqMan® Universal PCR Master Mix (Applied Biosystems). The reaction was performed on Applied Biosystems 7900HT Fast Real-Time PCR System, with standard mode thermal cycling conditions, according to the manufacturer’s instructions. Analysis of real-time PCR data to quantify gene expression level was done by comparative Ct methods [14].

Western blot

Following transfection (48 or 72 h) or exposure to Rubipy-SiO2 NPs (6 h), cells were rinsed twice with cold PBS and incubated with ice-cold lysis buffer (20 mM Tris–HCl, 100 mM NaCl and 1 mM EDTA, 0.5% Triton X-100) containing protease inhibitors cocktail (all from Sigma-Aldrich, Italy). After 10 min incubation on ice to ensure complete lysis, cell lysates were scrapped and centrifuged at 18,000×g for 15 min at 4 °C. The supernatant containing the cytoplasmatic protein fraction was transferred to a new tube. Protein concentration was measured by Bicinchoninic acid assay (BCA kit, Sigma-Aldrich, Italy). Equal amount of protein extracts (20 µg) were loaded onto a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Mini-PROTEAN® BIORAD). Separated proteins were transferred to a methanol-activated Hybond-P membrane (Amersham Biosciences, USA) (Mini Trans-Blot BIORAD®). The PVDF membrane was probed with a primary rabbit polyclonal antibody against clathrin heavy chain (Abcam, 1:1000), anti-caveolin-1 (Abcam, 1:800), anti-PAK1 (Prestige Antibodies, Sigma-Aldrich, 1:250), anti-SNX5 (Abcam, 1:1000) or anti-GAPDH (Millipore Cat MAB374, Italy, 1:7500) as loading control. The membrane was then incubated with the secondary anti-rabbit (Sanzta-Cruz, 1:5000) or anti-mouse (Zymax antibodies, 1:3000) antibodies IgG-horseradish peroxidase-conjugated and detected by enhanced chemiluminescence (ECL, Amersham Biosciences, USA).

Fluorescence microscopy

CaCo-2 cells were seeded at a density of 105 cells/well in 4-chamber slides (Falcon), grown for 24 h and left untreated or incubated with chlorpromazine 50 µM, dynasore 80 µM, methyl-beta-cyclodextrin 5 mM, nystatin 40 µg/ml, genistein 200 µM, or EIPA 75 µM for 1 h at 37 °C. To investigate the energy dependence of NP uptake, CaCo-2 cells were exposed to 200 μg/ml of 30 and 80 nm-sized fluorescent Rubipy-SiO2 NPs for 3 h at 37 or 4 °C in complete cell culture medium.

Following exposure, cells were washed 3 times in PBS, fixed with 4% (v/v) paraformaldehyde in PBS and permeabilised with 0.1% (v/v) Triton X-100 in PBS (Sigma-Aldrich, Italy) before staining with AlexaFluor 488-conjugated Phalloidin (Thermo Fisher Scientific, Italy), diluted 1:100 for 40 min at RT. The nuclei were counterstained with the Hoechst 33342 dye (Dako, Italy). After staining, the cells were washed in PBS and mounted for microscopy. Images were acquired with an Axiovert 200 M inverted microscope equipped with a ApoTome slide module and Axiovision 4.8 software (Carl Zeiss; Jena, Germany), using a 40×/1.0 objective lens.

Evaluation of cell metabolic activity (MTT assay)

Cells were grown in 96-well cell culture plates (Costar) until 75% confluent, exposed to Rubipy-SiO2 NPs for 48 h or to chemical inhibitors for 3.5 h and then washed in PBS. Cell viability was evaluated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide] (Sigma-Aldrich, Italy) added to the cells in fresh complete culture medium at a 250 μg/ml final concentration. After 2 h of incubation at 37 °C the supernatant was removed, the precipitated formazan crystals were dissolved in 0.1 M HCl in propan-2-ol and the absorbance was quantified at 540 nm in a multiwell plate reader (FluoStar, Omega, BMG Labtech, Offenburg, Germany). In parallel, to evaluate the possibility of interference of NPs with the assay, the PBS washing containing the silica NPs residues from each well was transferred to empty wells, incubated with MTT reagent in the conditions of the experiment and after 2 h the absorbance at 540 nm was read in a multiwell plate reader.

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Results

Characterization of the size distribution and agglomeration state of Rubipy-SiO2 NPs

Amorphous, negatively charged fluorescent Rubipy-SiO2 NPs of 30 and 80 nm were synthetized and characterized in water, PBS and cell culture medium as described previously [7]. The size distribution of Rubipy-SiO2 NPs in the complete CaCo-2 medium was measured by CLS immediately after preparing the NP suspension and after 24 h incubation at 37 °C (Fig. 1a; Table 1). In case of freshly prepared NP suspensions we observed a narrow size distribution of 80 nm NPs and a slightly larger peak of 30 nm NPs, indicating the initiation of the agglomeration already at this point. After 24 h incubation in the complete medium the size distribution has become much larger, and the average size of the particles similar for both types of Rubipy-SiO2 NPs. Moreover, visual inspection of both suspensions indicated agglomeration, and precipitation was visible to the naked eye.

📷Fig. 1

Size distribution of Rubipy-SiO2 NPs in complete cell culture medium. Rubipy-SiO2 NPs were suspended in CaCo-2 complete cell culture medium (10% of serum) at concentration of 1 mg/ml and the size distribution was evaluated either immediately or after 24 h pre-incubation at 37 °C by CLS (a) and by TEM (b)

Table 1

Average size and polydispersity of Rubipy-SiO2 NPs in water and complete cell culture medium measured by CLS (by weight), reported in nm

H2OComplete CaCo-2 medium (immediately)Complete CaCo-2 medium (24 h incubation)30 nm Rubipy-SiO2 NPs26.1 ± 1184.5 ± 64129 ± 9280 nm Rubipy-SiO2 NPs68.8 ±  2275  ± 49116 ± 114

The CLS results were confirmed by TEM images (Fig. 1b) showing in fresh suspensions well dispersed 80 nm NPs and the 30 nm NPs initiating to agglomerate, and in suspensions incubated for 24 h the agglomerates of both sizes of Rubipy-SiO2 NPs together with the clumps of the proteins.

Therefore, taking into account the difference in agglomeration state and in order to facilitate the comprehension of the manuscript we will refer to the NPs incubated for 24 h in the complete medium as “agglomerated”, and to the NPs freshly suspended in the complete medium as “non-agglomerated”, even if NP agglomeration had already started in freshly prepared suspensions, particularly in those of 30 nm.

Fluorescence characteristics of Rubipy-SiO2 NPs were described in our previous study [7]. To ensure that the agglomeration process is not interfering with fluorescence measurement we carried out a scan of the fluorescence emission of both types of Rubipy-SiO2 NPs freshly dispersed in the complete Caco-2 medium and after 24 h incubation at 37 °C. No major changes in the fluorescence signal were noted with the increase of agglomeration state (Additional file 1: Figure S1), however the background fluorescence of the cellular medium was interfering slightly with the measurements. Since the intensity of fluorescence emission of 80 nm NPs was higher than the one of 30 nm NPs at the same mass concentration, we performed a calibration curve of the fluorescence intensity versus the mass concentration for each size of Rubipy-SiO2 NPs (Additional file 1: Figure S2) and we calculated the reference values that enabled the comparison of the cellular uptake of both sizes of Rubipy-SiO2 NPs (Additional file 1: Table S1).

Cell uptake of agglomerated and non-agglomerated of Rubipy-SiO2 NPs

Rubipy-SiO2 NPs did not induce any toxicity to CaCo-2 cells for up to 48 h, as assessed by MTT assay (Additional file 1: Figure S3). For the quantification of the cellular uptake the cells were exposed to Rubipy-SiO2 NPs pre-incubated in the complete medium for 24 h and, in parallel, to Rubipy-SiO2 NPs freshly added to the complete medium, at the same concentration (200 µg/ml) and for the same exposure time (3 h). Measurements of the cell-associated fluorescence by flow cytometry, normalized to the fluorescence of NPs per mass unit, indicated a much higher cell uptake of the agglomerated form, particularly of 80 nm Rubipy-SiO2 NPs, than of the non-agglomerated form of NPs (Fig. 2).

📷Fig. 2

Effect of agglomeration state of Rubipy-SiO2 NPs on the level of cell uptake. The cellular uptake of Rubipy-SiO2 NPs was quantified by flow cytometry after 3 h exposure of CaCo-2 cells to 200 µg/ml of 30 and 80 nm Rubipy-SiO2 NPs either added to complete medium immediately before exposure (grey bars) or pre-incubated in complete medium for 24 h at 37 °C (black bars)

TEM images were obtained after 3 and 6 h exposure to Rubipy-SiO2 NPs and showed an increased presence of NPs inside the cells after 6 h comparing to 3 h exposure (not shown). Both the agglomerated and non-agglomerated forms of 30 nm and the agglomerated 80 nm Rubipy-SiO2 NPs were observed as clusters, either interacting with the cellular membrane or in big endosomal vacuoles inside the cells (Fig. 3), whereas non-agglomerated 80 nm NPs were observed as small groups or single particles, thus showing a difference in the agglomeration state also at that level. The presence of many large clusters of 80 nm agglomerated Rubipy-SiO2 NPs inside the cells was particularly striking. Beside the large vacuoles, NPs were also observed in early and late endosomes (sometimes interestingly in flask-shaped endosomes) and lysosomes. In many areas and under all conditions it was possible to see characteristic ruffles of the plasma membrane involved in the macropinocytotic process (Fig. 3) or smaller invaginations of the membrane associated with clathrin- or caveolae-mediated endocytosis.

📷Fig. 3

Cell uptake of Rubipy-SiO2 NPs observed by TEM. TEM images were obtained after 6 h exposure of CaCo-2 cells to 30 and 80 nm Rubipy-SiO2 NPs, either freshly added to cell medium or pre-incubated in the complete medium for 24 h (agglomerates); on the images we can observe clusters of NPs (black arrows), endosomes (E) and lysosomes (L) containing NPs, membrane ruffling typical for macropinocytosis (M), membrane invaginations (Asterisk). Note the flask-shaped endosomes on the right-bottom image (80 nm aggl)

The evaluation of the kinetics of cellular uptake of Rubipy-SiO2 NPs was performed after 1, 3 and 5 h exposure at 37 and at 4 °C using flow cytometry (Additional file 1: Figure S4A). At 37 °C the interaction of the agglomerated 80 nm NPs with the cells was very intense up to 3 h and then reached a plateau, suggesting saturation, whereas the cell uptake of 30 nm Rubipy-SiO2 NPs was increasing proportionally to the time of exposure. Experiments carried out at 4 °C showed a strong inhibition of the cell uptake of Rubipy-SiO2 NPs indicating that the mechanism of the internalization was energy-dependent (Additional file 1: Figure S4 A, B). Still, a slight cell-associated fluorescence was detected in these conditions, though not increasing in time, suggesting that NPs were interacting with the cell membrane also at 4 °C.

Effect of chemical inhibitors on cell uptake of Rubipy-SiO2 NPs , , , ,

. . . .

Effect of chemical inhibitors on cell uptake of Rubipy-SiO2 NPs

The use of chemical inhibition of the endocytic pathways, however widespread, is frequently criticised for lack of specificity, toxicity and multiple side effects [15, 16]. Consequently, a careful evaluation of the inhibitors’ effects on different endocytic pathways and for each tested cell line must be undertaken before actual experiments. Here, we used the classical markers of the endocytic pathways to assess the efficacy and the specificity of the employed inhibitors (Additional file 1: Figure S5): transferrin for clathrin-mediated endocytosis (CME), Bodipy-Lactosylceramide complexed to BSA (LaCer) for caveolae-mediated pathway and Dextran 10 kDa for macropinocytosis. We also evaluated the effect of the inhibitors on cellular morphology and on cell metabolism (Additional file 1: Figures S6, S7) to guarantee the absence of toxic effects on the cells in the range of the concentrations used in the study.

Fluorescence of the cells exposed to Rubipy-SiO2 NPs after the treatment with the endocytosis inhibitors was compared to the fluorescence of the cells not pre-treated with the inhibitors. The inhibitors of CME did not decrease the uptake of Rubipy-SiO2 NPs, except a slight but significant effect of chlorpromazine on the uptake of agglomerated NPs (Fig. 4). This inhibitor was shown to act strongly and specifically on the CME (Additional file 1: Figure S5). Methyl-β-cyclodextrin (MβCD), which is inducing cholesterol depletion in the lipid rafts, blocked the cell uptake of Rubipy-SiO2 NPs almost completely and was the most potent among the tested inhibitors. However, it is not a specific inhibitor, acting on both caveolae-mediated endocytosis and macropinocytosis pathway (Additional file 1: Figure S5). Nystatin, a specific inhibitor of caveolae-mediated pathway was more efficient for non-agglomerated form of NPs (both 30 and 80 nm, ~40–50% remaining cell uptake) than for their agglomerated form (~60% remaining cell uptake). The opposite effect was observed after the treatment with genistein, which reduced the uptake of agglomerated 30 nm Rubipy-SiO2 NPs by almost 80%, while of non-agglomerated form of these NPs only by around 20%. However, in our experiments with endocytosis markers we observed the lack of specificity of this inhibitor, since it was acting on all three tested pathways (Additional file 1: Figure S5). Similarly, also ethyl-isopropyl amiloride (EIPA), frequently used to inhibit macropinocytosis, was shown to be non-specific. In our study, EIPA demonstrated a higher efficacy in the reduction of cell uptake of 30 nm NPs than 80 nm NPs, no matter what their agglomeration state was (Fig. 4).

📷

Fig. 4

Inhibition of cell uptake of Rubipy-SiO2 NPs by endocytosis inhibitors. CaCo-2 cells were treated with the endocytosis inhibitors for 30 min, then exposed to 30 nm (a) and 80 nm (b) Rubipy-SiO2 NPs either freshly added to cell medium (grey bars) or pre-incubated in the complete medium for 24 h (black bars). The cellular uptake of NPs was quantified after 3 h exposure by flow cytometry and calculated as a percentage of the cell uptake in absence of the inhibitors, evaluated in parallel. Statistical significance was assessed by a Student’s t test. *p 80%) (Fig. 8a) the depletion of CAV1 at the protein level was not very successful (Fig. 8b) since the protein was still present in the transfected cells, probably due to efficient recycling. Consequently caveolae-mediated internalization of LaCer was only slightly reduced compared to non-silenced cells (Fig. 8c). We evaluated the uptake of Rubipy-SiO2 NPs by the cells transfected with si-cav1 in comparison to the cells transfected with si-ctrl. However, we could observe only a slight reduction in the level of cell-associated Rubipy-SiO2 NPs after 3 h exposure and after 6 h exposure (Fig. 8d, e), suggesting that the partial depletion of CAV1 was not sufficient to effectively inhibit the endocytosis of NPs.

📷

Fig. 8

Inhibition of cell uptake of Rubipy-SiO2 NPs in CAV1-silenced cells. CaCo-2 cells were transfected with si-ctrl or si-cav1 and after 48 h the remaining RNA expression was evaluated with real-time PCR (a), protein expression was assessed with western blot (b), the cell uptake of LaCer was measured after 30 min incubation (c) and the cell uptake of Rubipy-SiO2 NPs was measured after 3 h (d) and 6 h (e) exposure by flow cytometry. Statistical significance was assessed by a Student’s t test; *p 

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Review Open Access Published: 31 May 2021 Nanomaterials for cancer therapy: current progress and perspectives Zhe Cheng, Maoyu Li, Raja Dey & Yongheng Chen Journal of Hematology & Oncology volume 14, Article number: 85 (2021) Cite this article9 Citations 2 Altmetric Metricsdetails Abstract Cancer is a disease with complex pathological process. Current chemotherapy faces problems such as lack of specificity, cytotoxicity, induction of multi-drug resistance and stem-like cells growth. Nanomaterials are materials in the nanorange 1–100 nm which possess unique optical, magnetic, and electrical properties. Nanomaterials used in cancer therapy can be classified into several main categories. Targeting cancer cells, tumor microenvironment, and immune system, these nanomaterials have been modified for a wide range of cancer therapies to overcome toxicity and lack of specificity, enhance drug capacity as well as bioavailability. Although the number of studies has been increasing, the number of approved nano-drugs has not increased much over the years. To better improve clinical translation, further research is needed for targeted drug delivery by nano-carriers to reduce toxicity, enhance permeability and retention effects, and minimize the shielding effect of protein corona. This review summarizes novel nanomaterials fabricated in research and clinical use, discusses current limitations and obstacles that hinder the translation from research to clinical use, and provides suggestions for more efficient adoption of nanomaterials in cancer therapy. Background Despite significant advances in medical science and technology, cancer remains a disease with limited treatment approaches. Metastasis and recurrence of cancer contribute a lot to disability and mortality, and the exact mechanisms remain to be illuminated [1, 2]. Cancer is generally considered as the consequence of gene mutations [3]. In 2018, there were an estimated 18.1 million new cancer cases and 9.6 million deaths were caused by cancer [4]. According to the Global Cancer Observatory (GCO), approximately 30 million cancer patients will die from cancer each year by 2030 [5]. In addition to the high mortality of cancer, the economic burden on families of cancer patients and society is enormous. Therefore, efforts on cancer prevention, diagnosis and treatment are of great importance.Cancer is characterized by abnormalities in mechanisms that regulate cell cycle, leading to the survival and proliferation of malignant cancer cells. Signaling pathways are usually altered when cancer occurs. Inhibition of physiological apoptosis contributes to cancer development as well as resistance to radiotherapy and chemotherapy [6]. Inflammation and immune system disorder are also related to cancer. Traditional tumor staging (AJCC/UICC-TNM classification) is based on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N), and tumor metastases (M). Cancers can also be classified according to organs of origin, such as lung, colon, breast, head and neck, kidney, bladder, prostate, ovary, or various cancer cell types [7].Current cancer diagnosis approaches include imaging methods, laboratory tests, and morphological analysis of tissues and cells, which is usually considered highly reliable in most cancer diagnosis [8]. Pathological characteristics such as immunohistochemical (IHC) analysis, histological alterations, mutational and molecular genetics analysis also help cancer diagnosis [9]. Common cancer treatment consists of surgical resection, chemotherapy, radiotherapy, and biological therapy. Surgery is an effective measure to remove malignant solid tumors, especially in an early stage of cancer development. Combined therapy involves several therapies such as surgery, chemotherapy, and radiotherapy. The application of chemotherapy has been popular over the years for its simplicity and convenience in treating cancer patients [10, 11].Chemotherapy is effective for various cancers, including acute myelogenous leukemia, acute lymphoblastic, Hodgkin’s and non–Hodgkin’s lymphoma, small cell lung cancer, germ cell cancer, ovarian cancer and choriocarcinoma [12]. However, the indiscriminate cytotoxicity of chemotherapy causes undesirable side effects, as chemotherapy can also inhibit rapid-growing tissues and cells including hair follicles, gastrointestinal tract cells, and bone marrow. The use of chemotherapy also induces multi-drug resistance (MDR) and has potential association with cancer stem cells (CSCs). Cytotoxic chemical drugs used in chemotherapies are non-specific and heterogeneous in terms of distribution that contribute to MDR in the treatment process [13, 14]. This non-specificity impedes chemotherapy efficacy and impairs inhibition of tumor growth, metastasis and recurrence [15].Current chemotherapy faces problems such as lack of specificity, cytotoxicity, short half-life, poor solubility, occurrence of multi-drug resistance and stem-like cells growth. To overcome these disadvantages, nanomaterial-based chemotherapy, targeted therapy, molecular therapy, photodynamic therapy (PDT), photothermal therapy (PTT), chemodynamic therapy (CDT), and sonodynamic therapy (SDT) are being used in cancer treatment. In addition, a substantial number of studies on variety of therapies such as molecular therapy, apoptosis regulations, immunotherapy, signal modification therapy, nucleic-acid-based therapy, and anti-angiogenesis therapy for the treatment of cancer have been done in recent years [16,17,18]. With the advent of nanotechnology nanomedicines used in cancer therapy can possibly reduce drawbacks of chemotherapy and an extensive research studies have been going on along this direction. Nanotechnology applied in cancer therapy Properties of nanomaterials Medical nanotechnology uses materials with nanorange size, which is generally 1–100 nm. These materials are applied in the therapeutic drugs and devices design, manufacture [19]. As the size shrinks to nanoscale, many unique optical, magnetic and electrical properties emerge, making nanomaterials differ from traditional macromolecules. Typical nanomaterials possess several common characteristics: high surface-to-volume ratio, enhanced electrical conductivity, superparamagnetic behavior, spectral shift of optical absorption, and unique fluorescence properties. In the medical field, nanomaterials can be applied in drug transportation, controlled release. Increased permeability enabling crossing through biological barriers and improved biocompatibility are also noticeable features [20].These particular properties of nanomaterial suggest it can be utilized in cancer therapeutics. The high surface-to-volume ratio of some nanomaterials can assemble with biomolecules or residues, which can enhance the specificity of chemical drug complex in targeted therapy, thereby enhancing the efficacy of nanomaterial-based treatment while reducing its toxicity to normal cells [21]. PDT and PTT are two treatment methods related to optical interference. In PDT, a photosensitizer is accumulated in cancerous sites; when irradiated with certain wavelength light, singlet oxygen and other cytotoxic reactive oxygen materials are generated, causing apoptosis and/or necrosis [22]. PTT uses materials that possess high photothermal conversion efficiency to elevate the temperature of targeted cancerous areas, leading to cancer cell death. PDT and PTT are emerging cancer treatment methods with great potential, and materials used in these two therapies are under intensive research. Some nanomaterials can be used in PDT and PTT because of their unique fluorescence properties [23]. The superparamagnetic behavior of nanomaterials provides several usages for cancer diagnosis and treatment. A common nanomaterial, superparamagnetic iron oxide nanoparticles (SPION), has potential in cancer hyperthermia treatment due to its smaller size, higher targeting specificity, controllable releasing speed, and immune evasion capability [24].Progress of nanotechnology in targeted delivery Targeted delivery is one of a major advantage of nanomaterial-based cancer therapy over free drugs. Recent progress has been made in targeted delivery based on nanomaterials. The idea of targeted delivery aims for precise targeting of specific cancer cells, and it is achieved by either passive targeting or active targeting. Enhanced permeability and retention (EPR) effect is used in passive targeting while active targeting is achieved by conjugating with antibodies, peptides, aptamers and small molecules. Compared with free drugs, targeted delivery helps reduce toxicity in normal cells, protect drugs from degradation, increase half-life, loading capacity, solubility [19, 25].Through delicate design and modification, nano-drugs can maintain better specificity, bioavailability, less cytotoxicity to normal tissue, larger loading capacity, longer half-life period, and unique drug release patterns, overcoming disadvantages of conventional chemical therapy. During the past two decades, tremendous development in cancer pathology and nanoscience, technology, and industry (NSTI) created plenty of nanomaterials for cancer treatment and diagnosis.However, only a relatively small number of nano-drugs have been well developed and involved in clinical use. These nanomaterials can be generally classified into several categories (Fig. 1).Fig. 1📷Categories of nanomaterials applied in cancer treatment. a Nanoparticles. b Liposomes. c Solid lipid nanoparticles. d Nanostructured lipid carriers. e Nanoemulsions. f Dendrimers. g Graphene. h Metallic nanoparticles. PEG, poly(ethylene glycol)Full size image Nanomaterials used for cancer treatment Nanoparticles Polymeric nanoparticles Nanoparticles are particles with size of nanoscale. Polymeric nanoparticles (PNPs), mAb nanoparticles, extracellular vesicles (EVs), metallic nanoparticles are broadly researched nanoparticles (NPs) (Table 1). PNPs are defined as colloidal macromolecules with submicron size of 10–1000 nm. As drug carriers, PNPs carry chemical drugs and achieve the sustained release to targeted cancerous sites [26]. Drugs are encapsulated or attached to the surface of nanoparticles thus forming a nanocapsule or a nanosphere. The ingredients of nanoparticles have changed over the years. Initially, nonbiodegradable polymers such as polymethyl methacrylate (PMMA), polyacrylamide, polystyrene, and polyacrylates were used to fabricate nanoparticles [27, 28]. To avoid toxicity and chronic inflammation, polymeric nanoparticles made by these materials shall be cleared up in time. The accumulation of these types of polymer-based nanoparticles in tissues to a toxic level caused due to the difficulty to get them degraded, excreted, or physically removed have now been solved. Biodegradable polymers have been manufactured to reduce toxicity, improve drug release kinetic patterns and increase biocompatibility. These polymers include polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(amino acids) [29], poly(ε-caprolactone) (PCL), and natural polymers consist chitosan, alginate, gelatin and albumin. These improved polymeric nanoparticles have special advantages due to their properties and structures. For volatile pharmaceutical agents, PNPs help increase stability. For chemical drugs, PNPs provide optional administration methods such as oral and intravenous and higher loading ability compared to free drugs. The ability that protects drugs from degradation helps minimize undesired toxicity to normal tissues; for instance, PNPs loaded with cisplatin such as dexamethasone or α-tocopheryl succinate have been employed in chemotherapy, which prevents cisplatin-induced ototoxicity [30].Table 1 Summary of NPs in development or research stage for cancer therapyFull size tableThere are two main drug delivery methods: passive targeting and active targeting (Fig. 4a). A dense extracellular matrix causes difficulty for drugs to infiltrate while over-activated angiogenesis poses a certain advantage objectively known as EPR. When tumor grows, plenty of nutrition and oxygen are needed; in the meantime, tumor-induced angiogenesis generates many immature vasculatures that suppresses lymphatic drainage [39]. These leaky blood vessels make it possible for chemical drugs to penetrate into cancerous sites. However, the size of drugs is crucial as regular particles are not small enough to percolate through cancerous cells. On the contrary, nanoparticles and related chemical drug vehicles can easily penetrate targeted sites and accumulate because of attenuated lymphatic drainages [40].PNPs share the common property of high surface-to-volume ratio as nanoscale particles, making it convenient to attach targeting polymers onto the surface. A proven research has shown that bioavailability can be enhanced by coating polymers with polysorbates, utilizing polysorbates surfactant effect through endothelial cell membrane solubilization and fluidization. Surface coating helps PNPs interact with blood–brain barrier(BBB) endothelial cell membranes and facilitate endocytosis [41, 42]. As novel nanocarriers function differently from conventional chemical therapy, polymeric nanoparticles can deliver several sorts of chemicals to target sites including anti-cancer drugs, small interfering RNAs (siRNA), radionuclide, and specially designed polymeric nanoparticles possessing the ability to react to ultra-sound. Fluorescent polymeric nanoparticles are used as theragnostic tools. Theragnostic is a strategy combining diagnosis and treatment at the same time. Fluorescent polymeric nanoparticles (FNPs) have been identified as novel theragnostic materials in recent years. To achieve both diagnostic and therapeutic functions, nanomaterials with complex structures are fabricated. A FNP usually consists of fluorescent proteins, biocompatible biopolymers, inorganic quantum dots, and organic dyes [43]. In addition to tumorous imaging, drugs can be loaded by π–π bond or hydrophobic interactions in fluorescence assays that eventually enhances the anti-cancer efficacy of nanomedicine [44]. In siRNA delivery, cyclodextrin polymer (CDP)-based nanoparticles improve delivery efficacy in vivo [45]. Research studies have shown that transferrin modified adamantane-Polyethylene glycol (AD-PEG) and adamantane-PEG-transferrin (AD-PEG-Tf) are appropriate to deliver nucleic acid in vivo [32, 46]. Nanoparticles can be used to encapsule radionuclide such as I125 via electrophilic aromatic substitution which is in high radiochemical yields. Through this straightforward way, radionuclide can be stored in the stable core [47, 48]. Dey [49] developed a self-assembling peptide/protein nanoparticle with the size only 11 nm in diameter and it exhibited good biocompatibility and stability in vivo, indicating it should be suitable for drug delivery in cancer treatment. Ultrasound sensitive polymeric nanoparticles have emerged as an efficient tool for cancer diagnosis and treatment. Several uses of ultrasound interactive nanoparticles have been implemented. Use of ultrasound in NP manufacture helps enhance efficacy of drug delivery, therefore leads to reduction of side effects through improved traversing ability to overcome the barriers in cancer therapy. These barriers include endothelial blood vessels [50], tissue endothelium, interstitium, nuclear membrane and BBB [51, 52]. Since ultrasound can result in a thermal effect that may eventually break the nanoparticles, ultrasound can also be used as a preset trigger through which chemical drugs can be released under control [53]. However, the polymeric nanoparticle has its disadvantages: evidence shows that some polymeric nanoparticles undergo toxic degradation and toxic monomers aggregation thereby needing further studies for their improvement in fabrication and chemical properties [54].mAb nanoparticles Recent progress has been made in mAb nanoparticles. In targeted therapies, monoclonal antibodies (mAbs) are vastly used for their specific targeting ability and anti-tumor effect. Moreover, in recent years, mAbs are used in designing novel anti-tumor nanoplatforms and has been forefront in the field.To further increase therapeutic efficacy of anticancer drugs, mAbs are conjugated with cytotoxic drugs, this is termed as antibody–drug conjugates (ADCs); With specific antigens expressed differently in cancerous cells and normal cells guiding the drug complex, better specificity and less toxicity can be achieved [55]. Trastuzumab (Herceptin) is a mAb used to treat breast cancer with positive expression of human epidermal growth factor receptor 2 (HER2). Research using trastuzumab (Tmab) in ADC system have been conducted, and the result shows improved therapeutic efficacy compared with Tmab alone [56]. Abedin et al. fabricated an antibody–drug nanoparticle, which consists of a core loaded with paclitaxel (PTX) and a surface modified with trastuzumab. Two HER2-positive cell lines and one HER2-negative cell line were treated with this novel NP, PTX, and trastuzumab separately and the result was inspiring: NP complex showed better anti-tumor efficacy than PTX or trastuzumab alone, and relatively lower cytotoxicity in human breast epithelial cell control was observed in NP complex group [34] (Fig. 4a). Trastuzumab NPs based on ADC mechanism are promising nanoplatforms in cancer therapy and vast research are being conducted [57,58,59].Extracellular vesicles EVs are bilayer phospholipid vesicles with the size typically range from 50 to 1000 nm [60]. EVs are secreted continuously by various cell types and differ in size, origin, and content. Based on the origin, EVs are classified into three major groups: exosomes, microvesicles and apoptotic bodies [61, 62]. Exosomes are 40–200 nm nano-scale particles. EVs contain protein, RNA and DNA and are involved in long-distance communications [63]. Because exosome membrane contains similar lipids and molecules to their origin cells, exosome NPs can escape the immune surveillance and internalize smoothly with target cells, and exosome NPs are natural carriers to be combined with existing anti-tumor compositions and methods.Gene therapy utilizes DNAs/RNAs in cancer treatment to take effect. Several approaches are explored in gene therapy, including restoring mutated proto-oncogene such as p53 [64], inhibitor of growth 4 (ING4), phosphatase and tensin homolog (PTEN) [65] and gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) system that disables key oncogenes [66, 67]. RNA interference (RNAi) can be caused by small RNAs such as siRNAs and microRNAs (miRNAs). RNAi contributes to physiological and pathological process. Research that targeting oncogenic mRNAs by siRNA is under evaluation [68]. Gene therapy can also induce cell death by delivering transgene or cell death-triggering gene to tumor cells [69].Researchers have utilized exosomes as nanoparticle platforms to delivery nucleic acids, small molecules, and proteins [36, 70] (Table 2). Hadla et al. used exosomes loaded with DOX (exoDOX) to treat human breast cancer cells and the result showed that compared with free DOX, exoDOX enhances the cytotoxicity of doxorubicin and avoid drug accumulation in the heart [36]. Exosomes can be engineered for targeted delivery in cancer treatment. A macrophage-derived exosome was modified with aminoethylanisamide-polyethylene glycol (AA-PEG) moiety, and the AA-PEG exosome was loaded with PTX. The engineered exosome showed improved therapeutic outcomes in pulmonary metastases mouse model [71]. Jeong et al. utilized exosomes to deliver miRNA-497 (miR-497) into A549 cells, and the result showed that tumor growth as well as expression of associated genes were suppressed, indicating this exosome-mediated miRNA therapeutic can be used in targeted cancer therapy [72]. Compared with synthetic NPs, exosome NPs possess inherent biocompatibility, higher chemical stability and the ability to manage intercellular communications. However, there are obstacles of exosome NP application, such as lack of uniform criteria of exosomal isolation and purification, unclear mechanism of exosome in cancer treatment, heterogeneity and difficulty preserving [73,74,75].Table 2 EVs used as nanocarriers in cancer therapyFull size tableLipid-based nanomaterials Research on lipid-based nanomaterials is blooming and three main categories have been receiving great attention in current research and clinical trials: liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs). Liposomes were approved in 1965 and considered the first enclosed microscopic phospholipid bilayer nanosystem [81]. Liposomes are spherical vesicles composed mainly of uni-lamellar or multi-lamellar phospholipids, and the size of a liposome usually ranges from 20 nm to more than 1 um [82, 83]. A liposome generally has a hydrophilic core and a hydrophobic phospholipid bilayer. This kind of structure enables entrapment of both hydrophilic and hydrophobic drugs [84] depending on the pharmacokinetic properties of the drug. Liposomes with the typical structure encapsulate hydrophilic drugs within their aqueous core and hydrophobic drugs in the lipid bilayer. Drugs encapsulated within the central cavity of liposome are protected from environmental degradation during the circulation through human bloodstream [85]. The size and number of bilayers are two important parameters that affect the loading amount and half-life of drugs; therefore, liposomes can be classified into two types according to these two conditions: unilamellar vesicles and multilamellar vesicles (MLV). Unilamellar vesicles are further divided into small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV). An onion-like structure is formed in multilamellar liposomes, while several unilamellar vesicles can be formed inside other vesicles and form multilamellar concentric phospholipid spheres separated by water molecules [86].As per extensive research on nanocarriers, recent liposomes bear plenty of unique properties and characteristics; correspondingly, novel applications have emerged based on liposome materials. Three major issues have been discovered and dealt with over the development of liposomes. Breaking through biological barriers and avoidance of rapid clearance are problems researchers have been encountering. As referred to above, biological barriers have always been major technical obstacles for nanocarriers to overcome. Regarding liposomes, cells of the mononuclear phagocyte system (MPS) predominantly in the liver and spleen are acting as human guards and phagocytizing nanoliposomes. Modifying membrane is one of the major techniques to prolong liposome half-lives. Covering the membrane with proteins, peptides, polymers, or other molecules significantly enhances the ability to escape from the MPS system and therefore helps achieving longer liposomal half-lives [87]. This kind of liposomes was named “Stealth” liposomes. Later polyethylene glycol conjugated liposome was found to have a longer half-life compared to other modified liposomes. Based on this observation, PEG-liposomes loaded with doxorubicin (DOX) were used to treat Kaposi's sarcoma in HIV patients [88].Drug-loading and controlled release of liposomes are also important issues that need attention in liposome nanocarrier design. For cancer chemotherapy, bioavailability affects drug efficacy. Compared to free DOX, DOX liposome has a lower bioavailability, indicating that improving the bioavailability should be considered when design liposomes [89]. Co-delivery and controlled release are two major applications of liposomes. Combinations with chemical drugs, metals, gene agents, and other chemotherapeutic agents have been formed. Overactivation of certain signaling pathways is one of the patterns of cancer occurrence, and drugs targeting these signaling pathways are applied. To achieve higher efficacy, researchers loaded a novel PEGylated liposomal with ncl-240 and cobimetinib which are small-molecule inhibitors of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway and mitogen-activated protein kinase kinase/ extracellular signal regulated protein kinase (MEK/ERK) pathway, respectively. The result showed that the cytotoxic effect was enhanced due to synergistic effects [90]. A novel liposome-encapsulated nanocarrier loaded with both irinotecan and floxuridine showed better efficacy in advanced solid tumors [91]. The delicate structure of a novel liposome with multiple layers enabled it to effectively load up to 3500 siRNA molecules in a single bilayer and codelivery of DOX, which demonstrated better DOX efficacy and shrink of tumor mass in breast cancer treatment [92]. Triggered release and target methods are extensively studied. As cancerous areas have an average 6.8–7.0 extracellular pH value, which is slightly more acidic than healthy tissue [93], liposomes can be designed to release drugs when reaching acidic cancerous areas. With a pH-sensitive material, carboxymethyl chitosan (CMCS) coated to the surface, the cationic liposome (CL) preloaded with sorafenib (Sf) and siRNA (Si) obtained pH-sensitive property. Results showed that sorafenib release was enhanced and cellular uptake was increased at the pH of 6.5 [94]. Other than pH-responsive property, liposomes can also be fabricated with enzyme-responsive, redox-responsive, light-responsive characteristics, depending on tumor microenvironment (TME) and drug properties [95]. TME is the concept of the environment in which tumor cells are living. TME facilitates tumor growth, invasion, migration, angiogenesis, inflammation and it is related with drug resistance [96, 97]. The common characteristics of TME include the presence of EPR, hypoxia (lack of oxygen), acidosis (low pH), extensive angiogenesis, and tumor-associated immune cells that help the immune escape of cancer cells [98]. In general, liposomes' advantages are protecting loaded drugs from enzyme degradation, low toxicity, biocompatibility, flexibility, superior biodegradability, and non-immunogenicity [99]. However, application of liposome is limited due to disadvantages such as short shelf life, low encapsulation efficacy, dissatisfying stability, rapid removal by MPS, cell adsorption, and intermembrane transfer.SLNs are colloidal nanocarriers with a nanoscale of 1–100 nm. Because of the strict limits on the size, SLNs are referred to as the “zero-dimensional” nanomaterials, as they differ from other larger nanomaterials by at least one dimension in nanoscale. Unlike liposomes, the ingredients of SLNs include solid materials such as solid lipid, emulsifier, and water. Partial glycerides, triglycerides, fatty acids, waxes, steroids and PEGylated lipids are lipid used in SLNs [19]. In terms of structure and function, there are similarities and differences between SLNs and conventional liposomes. The similarities are the lipidic outer layer and delivery function of chemical drugs. Unlike traditional liposomes which consist of lipid bilayers that surround an aqueous pocket, some SLNs do not have a contiguous bilayer; instead a micelle-like structure is formed and drugs are encapsulated in a non-aqueous core [100]. Lipid components of SLNs are solid at body temperature, and SLNs have better stability and prolonged release than liposomes. However, SLNs have limitations that are unpredictable gelation tendency and inherent low incorporation rates because of their crystalline structure [101].NLC carrier was developed in the past two decades as an improved generation of both liposome and SLN. To improve stability and loading capacity while maintaining intrinsic protection function, biocompatibility, and non-immunogenicity, NLCs are designed as a system consisting of a core matrix loaded with both solid and liquid lipids. NLCs can be administrated through multiple methods: oral, parenteral, inhalational, and ocular. As many drug compounds used in cancer treatment are lipophilic, NLCs have gained lots of attention in recent years [102].Nanoemulsions Nanoemulsions (NE) are colloidal nanoparticles made of aqueous phase, emulsifying agents as well as oil [103]. The size of nanoemulsion ranges from 10 to 1000 nm. Nanoemulsions are widely used drug nanocarriers, usually solid spheres with amorphous and lipophilic surface that exhibit negative charge. As nanoemulsions are heterogeneous mixtures containing oil droplets in aqueous media, nanodroplets are distributed with small size, and three typical types of nanoemulsions can be formulated: (a) water in oil nanoemulsion system in which water is dispersed in an aqueous medium; (b) oil in water nanoemulsion system in which oil is dispersed in an aqueous medium; (c) bi-continuous nanoemulsion [103]. Nanoemulsions have several advantages over most lipid-based nanomaterials and nanoparticles: optical clarity, thermodynamic stability, large surface area, convenience in manufacture, biodegradability, and ideal drug release profile [104]. Membrane modified nanoemulsions have been extensively studied. Co-delivery by nanoemulsions is one of the methods to enhance bioavailability and drug efficacy. The test results of a NE drug carrier system loaded with spirulina polysaccharides and PTX showed that it could improve the anti-tumor effect of PTX by regulating immunity through Toll-like receptor 4/nuclear factor kappa B (TLR4/NF-κB) signaling pathways [105]. A nanoemulsion system consisting of temozolomide, rapamycin, and bevacizumab was established to treat advanced melanoma. Through parenteral administration, enhanced cytotoxicity against melanoma cells and improved inhibition of tumor relapse, migration and angiogenesis were observed in vitro human and mouse cell models [106](Fig. 4b).Nanoemulsions can also be applied to immune therapy by loading certain immune-stimulation moiety. Cytokine Interferon gamma (IFN-γ) was loaded in a modified nanoemulsion to stay stable in extreme temperature changes for three months. The test results showed that this NE suppressed cell viability of MCF-7 human breast cancer cells and induced cellular activity of phagocytes, suggesting a promising potential in cancer treatment [107] (Fig. 4c). One application that gains plenty of attention is using NE as a strategy to overcome MDR. In MDR cancer cells, ATP-binding cassette transporters (ABCs) are responsible for part of MDR occurrence. MDR transporters expressed by ABCs cause resistance to anticancer drugs. P-glycoprotein (P-gp) is the first identified ABC transporter encoded by ABC1 gene which possesses function of pumping colchicine, vinblastine, etoposide and paclitaxel (PCX) from the cell [13]. To overcome this obstacle, a novel NE co-delivering baicalein and paclitaxel was fabricated by Meng and colleagues. By co-encapsulating these two drugs, oxidative stress was elevated, thereby providing a suitable strategy to improve cell sensitivity to paclitaxel. Results showed that reactive oxygen species (ROS) was increased, cellular glutathione (GSH) was decreased, caspase-3 activity was enhanced in MCF-7/Tax cells, and an in-vivo study showed that baicalein-paclitaxel NE exhibited a superior antitumor efficacy than conventional paclitaxel formulations [108, 109]. These studies clearly exhibit the potential benefit of using specially manufactured NEs in MDR management.Despite potential benefit NEs possess, there are challenges to clinical application. The production of NEs usually involves high temperature and pressure. Therefore, not all starting materials are suitable in NE application. This is one of the obstacles in applying NEs to massive commercial production. In NE preparation, high-energy methods such as homogenizer and microfluidizer are used, which makes NE costlier than other conventional formulation. Because of lack of understanding of chemistry in NE production, detailed research should be conducted about component interaction and NE metabolism in human body to assess the safety in clinical use [104].Dendrimers Dendrimers are a kind of unique macromolecules with hyperbranched defined architecture. The most apparent characteristic of dendrimers is their highly branched and easily modifiable surfaces. The size of these dendrimer polymers is ranging mainly from 1 to 10 nm, while some specially fabricated large dendrimers can reach up to diameters of 14–15 nm [110, 111]. Three major structural parts form the dendrimer molecules: central core that loads theragnostic agents through noncovalent encapsulation, branches that form the interior dendritic structure, and the exterior surface conjugated with functional surface groups. Several dendrimers have been developed for cancer therapeutics: polyamidoamine (PAMAM), PPI (polypropylenimine), PEG (poly(ethylene glycol)), Bis-MPA (2,2-bis(hydroxymethyl) propionic acid), 5-ALA (5-aminolevulinic acid), and TEA (triethanolamine) [112].Due to specific structure, dendrimers have unique features over other nanomaterials: defined molecular weight, versatile adjustable branches, narrow polydispersity index, superior solubility and bioavailability of hydrophobic drugs. Cationic dendrimers with positively charged surfaces can form complexes with nucleic acids; therefore, dendrimers can be used as efficient nucleic acid nanocarriers. PAMAM and PPI are two widely studied dendrimers with various application strategies. A PAMAM dendrimer/carbon dot nanohybrid was designed to achieve MDR management and cancer cell monitor simultaneously via fluorescence imaging. Two complexes were manufactured separately. The first part was a CDs/DOX complex consisting of blue-emitting carbon dots (CDs) and anticancer drug DOX through non-covalent interactions. The other part was G5-RGD-TPGS, which consists of generation 5 (G5) PAMAM dendrimers, targeting ligand cyclic arginine-glycine-aspartic (RGD) peptide and drug efflux inhibitor d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS). Two parts were connected by electrostatic interaction and formed a dual drug-loaded nanohybrid system. In vitro fluorescence was achieved by the luminescence of CDs, and targeting specificity was achieved by the presence of RGD ligands that targets αvβ3 integrin receptors overexpressed in cancer cells [113]. The results showed that TPGS had a significant inhibitory effect on cancer cells. The Co-delivery ability of dendrimer can also be used in delivering completely different materials. DOX is commonly used to treat colon cancers. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a crucial factor in the apoptotic pathway, capable of binding to death receptors 4 and 5 (DR4 and DR5), which are overexpressed in various cancer cells. Pishavar group encapsulated DOX and TRAIL plasmid in a dendrimer nanocarrier, which exhibited a stronger antitumor effect than modified carriers containing DOX or TRAIL plasmid alone [114]. A PAMAN nanocarrier based on dendrimer was synthesized and used for chemotherapy combined with photothermal treatment of liver cancer cells. Though PAMAN dendrimers without modification have disadvantages such as low transfection efficiency, inefficient cellular internalization and instability of encapsulation [115], the nanomaterial has competitive contrast properties, showing great potential in combination therapy [116].Carbon nanomaterials Carbon nanomaterials (CNMs) are a kind of nanosized material with many categories based on carbon element. CNMs have been widely used in many industrial and medical fields because of their unique electronic, thermal, optical, and mechanical properties (Table 3). In cancer theragnostic applications, CNMs are considered more biocompatible and safer than metal-based nanomaterials [117, 118]. CNMs can load chemical drugs through π–π stacking or hydrophobic interactions due to inherent hydrophobic nature, making CNMs as efficient drug delivery platforms [119, 120]. Several carbon nanomaterials have been massively studied in cancer treatment: graphenes, fullerenes, carbon nanotubes (CNTs), carbon nanohorns (CNHs), carbon quantum dots (CQDs) and graphyne (GDY). Although all these materials are based on carbon elements, the morphological structure, properties, and functions of these nanomaterials vary greatly.Table 3 Recent studies on CNMs for cancer therapyFull size tableGraphene is a two-dimensional crystal with sp2 -hybridized carbon sheet which possesses remarkable mechanical and electronic properties. It has also been heavily researched in biomedical applications, including cancer treatment [121]. Graphene-based nanomaterials can be classified into several types depending on their composition, structure, and properties: single-layer graphene, multi-layer graphene, graphene oxide (GO) and reduced graphene oxide (rGO) [122]. Graphene has unique electrochemical and mechanical properties, for example, optical transmittance, chemical inertness, high density, molecular barrier abilities and high hydrophobicity [123, 124]. Graphene also has other remarkable features that contribute to cancer theragnostic such as high planar surface enabling higher drug-loading capacity [125] and thermal conductivity (5000 W/mK) [126]. However, van der Waals forces and π–π stacking interactions cause poor solubility and agglomeration of nanosheets formed by graphene in solution, which significantly affects toxicity and hampers its fabrication [127, 128]. These drawbacks have driven researchers to look for more bioavailable graphene-based nanomaterials that retain graphene’s advantages while being easy to fabricate. GO is a chemically modified material based on graphene. Functional oxygen groups such as carboxyl (–COOH) and Hydroxyl (C–OH) locate at the edge of graphene, while carbonyl (C=O) and epoxy groups (C–O–C) locate on the basal plane, thereby forming a typical GO molecule [125]. A rGO is the reduced derivative of GO. Compared to graphene, GO and rGO have improved properties regarding to biological usage. Defective oxygen-bound sp3 carbon atoms exhibit strong hydrophilicity and help forming of dispersions in aqueous solvents that are highly stable colloidal, preventing uncontrolled van der Waals, hydrophobic interaction induced aggregation [129]. Meanwhile, hydrophilic functional groups on the GO surface make the nanosheets a versatile platform for conjugating of various materials, which provides great potential in targeted therapy, PDT, PTT, and cancer diagnosis [130, 131].Compared to other nanomaterials, graphene shows direct immunogenicity toward the immune system, and lateral size can regulate the extent of immunostimulatory capability both in vitro and in vivo [132]. Research shows that graphene activates the main components of the human immune system, macrophages and dendritic cells, indicating its potential in cancer treatment. Feito et al. studied the effect of the GO nanosheets specifically designed for hyperthermia cancer therapy on macrophage and lymphocyte function. The result showed that the 6-armed GO (6-GOs) significantly increased secretion of tumor necrosis factor alpha (TNF-α) by RAW-264.7 macrophages without changing IL-6 and IL-1β levels. In the presence of concanavalin A, lipopolysaccharide and anti-CD3 antibody, treatment of primary splenocytes involved 1-GOs and 6-GOs leading to significant dose-dependent cell proliferation and a decreased IL-6 level, which suggested the inherent weak inflammatory properties of GOs that are favorable for hyperthermia cancer therapy [133]. Graphene has also been found to inhibit some tumor cells. Burnett [134] treated human osteosarcoma (OS) cell and normal osteoblast cell with GO, and found that the apoptosis rate of OS cells was significantly higher than that of hFOB1.19 normal osteoblast cells. GO showed significant effects on cytotoxicity against OS, Nrf-2 decrease, ROS and cytomorphological changes. CSCs are generally considered a cancer cell population of high tumorigenic potency with self-renewable ability. CSCs interact with the TME and are believed to be involved in MDR formation [135]. Destruction of CSCs is one of the therapeutic approaches to avoid malignancy. It has been claimed that GO can specifically target CSCs rather than normal cells, and by inhibiting several key signaling pathways including WNT, Notch and STAT-signaling, GO induces CSC differentiation and inhibits tumor-sphere formation in multiple cell lines including breast, ovarian, prostate, lung, pancreatic and glioblastoma [136]. The researchers named this phenomenon, “differentiation-based nano-therapy”. However, few studies have been conducted over the past years, and more evidence may be needed. The interaction of graphene-immune cell interaction, effect graphene casts upon immune system and the direct anti-CSC phenomenon require further research.As a nanomaterial with a high surface-to-volume ratio and plenty of oxygen-containing branches, graphene is a suitable platform for drug delivery, PDT, PTT. A GO-peptide hybrid was fabricated via irreversible physical adsorption of the Ac-(GHHPH)4-NH2 peptide sequence known to mimic the anti-angiogenic domain of histidine-proline-rich glycoprotein (HPRG). The hybrid nanomaterial was tested in prostate cancer cells (PC-3), human neuroblastoma (SH-SY5Y), and human retinal endothelial cells (primary HREC). The results showed that this GO-peptide nanoassembly effectively induced toxicity in the prostate cancer cells, blocked the cell migration, and inhibited prostaglandin-mediated inflammation in PC-3 and HRECs. Since poor nucleation, internalization of liposomal doxorubicin (L-DOX) limited its application in breast cancer, a novel DOX-loaded GO nanocarrier was created. The GO-DOX exhibited much higher anticancer activities when administered to cellular models of breast cancer. Through live-cell confocal imaging and fluorescence lifetime imaging microscopy, researchers found that GO-DOX achieved its high efficacy by inducing massive intracellular DOX release when bonded to the cell plasma membrane [137]. Many research indicates that GOs and rGOs can target at hypoxia [138] and abnormal angiogenesis in cancer TME [139, 140]. GOs and rGOs are also widely used in PDT and PTT [141, 142]. GDY is an allotrope of graphene that contains two acetylenic linkages in each unit cell, which double the length of the carbon chains connecting the hexagonal rings [143]. As a result, GYD is softer than either graphyne or graphene. In the past three years, several studies have been conducted using GYD as a drug delivery platform for photothermal/chemotherapy combinatorial approach in cancer diagnosis [144,145,146].Fullerenes are molecules composed of carbon allotropes. The conformation of fullerenes includes hollow sphere, ellipsoid, or tube. Typical fullerenes include C60, C70, C82, etc. Metal atoms can be incorporated inside and form a metallofullerene [117]. Metal atoms encapsulated in the fullerene are usually Group III transition elements or a lanthanide. Since electrons of the intra-fullerene can transfer from encapsulated metal atom to the fullerene cage, metallofullerenes can be used as magnetic resonance imaging material. Properties of fullerenes also include free radical scavenging ability; therefore fullerenes can act as antioxidants [147, 148]. Compared to other nanomaterials, fullerene shows extraordinary properties in PDT and PTT. Chen et al. demonstrated that two critical factors leading to errors in photothermal efficiency estimation were laser irradiation time and nanoparticle concentration, and determined that photothermal conversion efficiency of polyhydroxy fullerenes was 69% [149]. The facts that the photothermal response of fullerenes remained stable with repeated laser irradiation, and the fullerene structure did not change, indicated that fullerenes were ideal candidates for photothermal therapy. A near-infrared (NIR) light-harvesting fullerene-based nanoparticles (DAF NPs) was tested for photoacoustic (PA) imaging-guided synergetic tumor photothermal and PDT. Compared to fullerene and antenna nanoparticles (DA NPs), DAF NPs showed better reactive oxygen species and heat generation efficacy. In vitro and in vivo studies demonstrated that DAF NPs could effectively inhibit tumor growth through synergetic PDT and PTT [150]. As a nanocarrier, fullerene has also been used in chemical drug delivery combined with PDT or PTT [150, 151].CNTs are cylindrical tubes formed by sp2 -hybridized carbon atoms considered as rolls of graphene. The size of CNTs can vary from 1 nm to several micrometers. According to the number of layers formed in a CNT, CNTs can be divided into single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). Poor water solubility and toxicity are two drawbacks of CNTs. Many studies on surface functionalization and material modifications have been carried out to solve the above problems and make CNTs more bioavailable. As a carbon-based nanomaterial, CNTs can interact with immune cells and induce immune response, therefore elevate immunity to suppress tumor growth [152, 153]. As a nanocarrier with a long research history being researched, CNTs are commonly considered an efficient PDT and PTT vehicle. Sundaram and his co-workers [154] coupled SWCNTs with hyaluronic acid (HA) and chlorin e6 (Ce6), and test this novel material in colon cancer cells using PDT. After 24 h, cellular changes were observed via microscopy, LDH cytotoxicity assay, and cell death induction. The result showed that the synthesized material enhanced the ability of PDT. Another synthesized NIR active photothermal agent, CNTs-PAMAM-Ag2S, was found to be highly efficient in PTT. The experiment showed that under irritation with 980 nm laser, photothermal efficacy of this complex was higher than that of copper-based and popular gold photothermal agents. Moreover, the complex demonstrated excellent stability against photo-bleaching and photo-corrosiveness, indicating the novel nanoagent could be promising in PTT [155].Drug delivery systems (DDSs) based on CNTs loaded with DOX, PTX [156], cis‑platinum (CDDP) have been intensively studied [157,158,159]. CNHs belong to the carbon allotrope family. The conical structure is usually between 2 and 5 nm in diameter and the length of the larger spherical superstructures forming with sp2 hybridized carbon atoms is typically around 100 nm, which partly resembles the CNTs [160]. Similar to CNTs, CNHs lack solubility and require surface modification to be a nanocarrier in human tissue. Solutions include adding organic species onto the outer skeleton [161], conjugate planar aromatic molecules through electrostatic association or π–π stacking interactions [162, 163]. CNHs possess both drug-loading and photothermal abilities and were used in design of DDS with combined characteristics. Yang et al. made a dual chemo drug-loaded single-walled CNHs system. SWNHs were modified with poly and mPEG-PLA via hydrophobic-hydrophobic and π–π stacking interactions. Cisplatin and DOX were loaded onto modified nanohorns separately. The nanocarrier exhibited loading ability and efficient photothermal ability with a pH-dependent releasing capacity. Results showed that both primary breast tumors and the lung metastases were eradicated [164]. CNHs can also be modified with specific targeting molecules and applied in target chemical therapy. A cisplatin loaded CNH attached with a mAb D2B, selective for prostate specific membrane antigen (PSMA) + prostate cancer cells, showed superior efficacy and specificity to kill PSMA + prostate cancer cells compared to hybrids Ab-CNHs and cisplatin-CNHs [165].Toxicity and side effects of CNMs used in cancer therapy have been studied in depth. Serum protein adsorption, hemolysis, cytotoxicity, and immunotoxicity have been reported for GO and rGO (93). As GO and rGO have a large surface area, they can be substrates for protein adsorption in the biological environment [176, 177]. With proteins adsorbed to the nanomaterial, loss of designed function and blockage of blood capillary might occur. In vitro and animal experiments indicated that the dose and size of GO and rGO could affect the toxicity of nanomaterials [178]. One study showed that large amounts hydrophobic rGO, accumulated on cell membrane, could induce high ROS stress and eventually lead to cell apoptosis [179]. In vivo studies revealed that CNTs could elicit chronic inflammation, granuloma formation, fibrosis, along with mesothelioma-like pathology [180]. Yan et al. summarized factors influencing CNT-induced toxicity such as surface modification, degree of aggregation, concentration, CNT size and shape, and listed up sites of CNTs accumulation after separation from anticancer drugs, which eventually suffer from CNT toxicity [181]. However, among this vast evidence achieved from various cells and animals, which aspect of CNMs plays a central role and the exact mechanisms of cellular toxicity caused by CNMs remain to be addressed [182].Quantum dots Quantum dots are widely researched biomedical imaging probes due to their distinctive optical and electronic characteristics. They are typically nanometer-scale semiconductor crystallites and are broadly used to improve the efficacy of fluorescent markers in biological imaging [183]. Compared with organic fluorophores, QDs possess unique optical and electronic properties such as size and composition leading tunable fluorescence emission from visible to infrared wavelengths, large absorption coefficients, and high brightness levels photostability [184]. There are three common QDs based on carbon: graphene quantum dots (GQDs), nanodiamond and CDs. The most common use of carbon QDs is bioimaging, which can be applied to cancer imaging and sensing. GQDs are considered emerging nanomaterials in biosensing and cancer therapy because of the inherent grand surface suitable for molecular conjugation, superior biocompatibility, and rapid excretion. A photoluminescent glycodendrimer with terminal β-cyclodextrin molecules system was designed and used for DOX delivery with biocompatibility and pH-sensitivity. GQDs were used to provide the surface for PAMAM to grow from. After excitation at 365 nm by UV light, the emission spectra from GQDs and GQDs-PAMAM-β-CD were recorded. The result showed higher efficiency in killing cancer cells than that achieved by DOX alone and containing the GQDs made it a potential imaging agent with photoluminescent activity [185].The fluorescent ability of GQDs was also used in a novel nanocarrier for targeted therapy. Researchers conjugated folic acid to sulfur-doped graphene quantum dots (FA-SGQDs) through simple pyrolysis of citric acid (CA), FA and 3-mercaptopropionic acid (MPA). The complex exhibited blue fluorescence with an emission band at 455 nm upon excitation at 370-nm wavelength, and a non-immunogenic FR-mediated endocytosis process for TA-SGQDs to enter the FR-positive cancer cells was revealed. In addition to bioimaging and biosensing, GQDs were also being investigated for PTT and PDT. A specifically modified GQD which exhibited strong absorption (1070 nm) in NIR-II region was prepared. The so-called 9T-GQDs having uniform size distribution, tunable fluorescence, and high photothermal conversion efficacy (33.45%) made it effective for ablating tumor cells and thus inhibited tumor growth under NIR-II irradiation, showing the potentiality of GQDs in PTT [186]. A combined photodynamic-chemotherapy DDS was designed based on carbon quantum dots. Researchers conjugated 5-aminolevulinic acid (5-ALA) with mono-(5-BOC-protected-glutamine-6-deoxy) β-cyclodextrin (CQD-glu-β-CD) moiety, and these materials were conjugated to CQDs loaded with DOX. High cytotoxicity and morphological changes of MCF-7 cancer cells were observed; also, ROS were induced by 15 min 635 nm (25 mW cm−2) radiation and achieved higher therapeutic effects [187]. CDs and nanodiamond have also been studied in cancer treatment utilizing its function of targeted therapy [188,189,190], PDT [191], cancer imaging [192] and antitumor immunity mediation [193, 194]. Compared with other carbon materials, research on carbon QDs is in its rising stage. Major obstacles in clinical translation of QDs are lack of standard protocol in high-quality QD production and their exact reaction mechanism and formation process [195].Metallic and magnetic nanomaterials Metallic nanoparticles have been extensively studied in bio-imaging and drug delivery because of their distinct optical, magnetic, and photothermal features. Metallic materials can be used in many forms in conjugation with versatile carriers such as NPs, liposomes, dendrimers or CNMs. Magnetic nanomaterials are mainly applied in MRI imaging. Guided by external magnetic field, magnetic NPs loaded with chemical drugs can target cancer cells, and therefore side effects of conventional chemotherapy are reduced [196] (Fig. 3). With metal particle conjugated, the nanosystem possesses both bio-imaging and PTT function. Iron oxide nanoparticles (IONPs) consisting of Fe3O4/Ag were encapsulated with a gold shell. MRI contrast capability was showed from IONPs and PTT due to the gold shell in the NIR region [37]. In cancer treatment, metallic materials are widely used in PTT, PDT, CDT, and immunotherapy. CDT is a Fenton or Fenton-like reaction-based therapeutic modality that relies on nanocatalyst [197]. Similar to PDT, highly oxidative hydroxyl radicals (·OH) are produced and toxic ·OH radicals take effect in cancer cells by triggering chain reactions with surrounding organic molecules, eventually leading to irreversible damage to DNA, lipids, and proteins [198]. During the process, iron-based nanostructures including FeS2, Fe2P, Fe3O4, SnFe2O4, and amorphous iron are used to catalyze disproportionation of H2O2 to generate ·OH radicals [38, 199, 200]. For PTT and PDT, as NIR possesses much stronger tissue penetration ability than ultraviolet (UV) and visible light, NIR triggered materials are crucial in these therapies. In PTT, cancer cells are eliminated to the generation of thermal energy, while ROS including ·OH, singlet oxygen (1O2), and superoxide (O2 − ·) induce cytotoxic reactions in PDT [201]. Au (gold), Cu (copper), Fe (iron) are commonly used metallic materials in these therapies [202,203,204] (Fig. 2). The disadvantage of metallic nanomaterials lies in their toxicity. Attarilar et al. summarized the mechanisms of metallic NPs: ROS generation and influence on cell structures, characteristics of metallic NP toxicity are similar to other NPs, that toxicity is related to size, shape, dimensionality, surface charge [205]. Therefore, metallic NPs should be carefully examined before use on human patients.Fig. 2📷Schematic illustration of nanomaterial involved PTT, CDT, PDT. With NIR irradiation, PTT materials such as GO/rGO generate heat and cause cancer cell death. CDT material BSA-CuFeS2 and specific wavelength light irradiated PDT material CNTs generate ·OH, 1O2, O2 − · from O2, H2O2 in cells and cause cancer cell death. CDT, chemodynamic therapy; CNT: carbon nanotube; GO: graphene oxide; NIR: near-infrared; PDT: photodynamic therapy; PTT, photothermal therapy; rGO: reduced graphene oxideFull size image Cancer treatment and nanomaterial design Approaches in cancer treatment To date, several mainstream approaches toward cancer treatment have been broadly applied to tackle cancer. Moreover, despite differences in working platforms, functional ingredients, and mechanisms, most researchers adopt two main targets: tumor cells and TME which include the immune system related to the tumor (Fig. 3).Fig. 3📷Illustration of interaction between nanomaterials and tumor cells. a Antigen–antibody conjugation modified nanoparticle endocytosis and transcytosis; b Liposome reaches cancerous area from blood vessels through EPR effect. c The magnetic nanoparticle coated with chitosan carries 5-Fluorouracil. Under external magnetic field, the nanoparticle shows passive targeting ability at cancer cells. d Therapeutic AuNP is blocked by BBB under normal status. After FUS exposure, the BBB is opened temporarily by microbubble inertial or stable cavitation and allows AuNPs to get through. BBB: blood–brain barrier; EPR: enhanced permeability and retention; FUS: focused ultrasoundFull size imageStrategies targeting cancer cells Targeting cancer cells is a natural method to eliminate cancer. With EPR and active targeting, modified nanocarriers such as NPs, dendrimers, or CNMs can reach cancer cells and release chemical drugs or biomaterials [206, 207]. Antibodies targeting specific antigens overexpressed on cancer cell surfaces are widely used in these platforms. After endocytosis by cancer cells, encapsulated chemical drugs exert cytotoxicity or nucleic acid materials induce cell apoptosis, depending on the encapsulated cargo. Progress has been made in nucleic acid delivery and nano-DDS based on exosomes [72, 78], PNPs, liposomes [208], dendrimers [115] are massively researched in cancer therapy.Strategies targeting TME Another strategy is about the TME that contain tumor cells. As mentioned above, angiogenesis is extremely active in almost all tumors because of uncontrolled cell proliferation and massive energy is needed for that. Research on this specific characteristic showed promising results. Sengupta designed a NP system specifically targeting abnormal tumor angiogenesis with combretastatin, and this medicine was co-encapsulated into the PLGA core with DOX. As a result, the DOX was efficiently taken up by the tumor after a rapid shutdown of the cancerous vessels induced by combretastatin, and an improved overall therapeutic index was achieved along with reduced toxicity [209]. In addition to abnormal vasculature, extracellular matrix (ECM) has also been researched in cancer treatment. ECM acts as a guiding scaffold in cancer proliferation, migration, invasion and angiogenesis [210]. Several main materials contributing to these cancerous properties are collagen, HA, various enzymes. As the main structural protein of the ECM, collagen forms migration tracks for tumor cells, while HA contributes to high interstitial fluid pressure (IFP), preventing drug diffusion and penetration [211, 212]. Enzymes, for example, matrix metalloproteinases (MMPs), can regulate TME by manipulating the activity of non-ECM molecules, including growth factors, receptors, and cytokines [213]. In nanocarrier design, ECM is one of the factors to be considered. Combined with conventional chemical drugs, recombinant human hyaluronidase (PEGPH20) in PEGylated form that targets at ECM hyaluronic acid exerted therapeutic effects for metastatic pancreatic cancer patients, especially in those with high hyaluronidase expression [214]. Efforts have been made to enhance chemical drugs loaded nanocarrier penetration ability in solid tumors by coating carriers with hyaluronidase (HAase) (Fig. 4b). This simple but effective strategy shows better anti-tumor efficacy [215].Fig. 4📷Cancer treatment approaches based on nanomaterials. a Targeting cancer cells by passive targeting or active targeting. b Targeting TME including anti-angiogenesis, stromal cell and extracellular matrix. Bevacizumab was loaded in liposome and conjugated with VEGF to inhibit angiogenesis. HAase was modified onto NP surface and enhanced NP penetration ability. c IFN-γ as an immune modulator delivered by liposomes activated immune cells in cancer immunotherapy. HAase: hyaluronidase; IFN-γ: Cytokine Interferon gamma; NP: nanoparticle; TME: tumor microenvironment; VEGF: vascular endothelial growth factorFull size imageNanomaterials and cancer immunotherapy The immune system plays a vital role in cancer formation and progression. There are several approaches in immunotherapy including immune checkpoint blockade therapy, chimeric antigen receptor (CAR)-T cell therapy, cancer vaccine therapy and immune system modulator therapy [216]. In these cancer immunotherapies, natural molecules or synthetic molecules are used to enhance or restore immune system function and exert anti-tumor effect. Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are important immune checkpoints and immune check point inhibitors (ICIs) targeting PD-1/PD-L1 has been researched to be loaded in nanocarriers targeting cancer [217]. In a research conducted by Bu and colleges, over-expression of PD-1 was considered to allow cancer cells to perform antitumor immunity evasion, and traditional immune checkpoint inhibitors (ICIs) of PD-1/PD-L1 showed inconsistent benefits. To ensure bonding of PD-L1 and ICIs, multivalent poly (amidoamine) dendrimers were employed; as a result, PD-L1 blockade effect was improved, and tumor site drug accumulation was enhanced [218]. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) is as an immune checkpoint with the function to downregulate immune responses [219]. Among these molecules are antibodies, small molecular inhibitors, proteins. Nanomaterials play an important role as drug vehicles to deliver these moieties (Table 4). Through these strategies, novel nanoplatforms can be developed and might achieve better efficacy and bioavailability than conventional therapies (Fig. 4) [106, 134].Table 4 Nanomaterials applied in cancer immunotherapyFull size tableAdvantages and challenges of nanomaterial applications in cancer therapy Nanomaterials applied in cancer therapy have advantages over conventional chemical drugs as well as challenges in application. Several significant hallmarks in tumorigenesis and tumor development have been elucidated: continuous proliferative signaling, growth suppressors evasion, cell death resistance, replicative immortality, induced angiogenesis, activating invasion and metastasis, inflammation, genomic instability, and mutation [224, 225]. Traditional chemotherapy and radiotherapy have disadvantages in efficacy and side effects because of unspecific distribution and indiscriminate cytotoxicity to cancer cells and normal cells. Therefore, a delicate balance of dosing and an advanced targeting DDS is of great importance in cancer treatment [226]. To reach cancerous target sites, chemical drugs taken orally or intravenously shall pass several “fortifications”: TME and vasculature, MPS, BBB and kidney filtration. In physiological conditions, barriers like normal tissue microenvironment, vasculature, RES, BBB, and kidney filtration contribute significantly to pathogen resistance. However, in cancer treatment, intake of anticancer chemical drugs is affected by these defenses. Cancer cells hold a different proliferation pattern than normal cells. Cancer tissues exhibit distinctly in the dense extracellular matrix, over-activated angiogenesis induced by excessive angiogenic factors and high interstitial fluid.Nanomaterial and drug metabolism Drug metabolism is a complex process. MPS, also called as reticuloendothelial system or macrophage system [227], consists of blood monocytes, tissue macrophages, and other immune cells. When dealing with extrinsic molecules, in this case, chemical drugs, parts of the MPS such as immune cells in the liver, spleen, or lungs will react, and activated macrophages or leukocytes quickly eliminate the drugs, causing short drug half-life [228]. Nanocarriers with surface modification such as PEG or specific peptide possess lower MPS clearance and therefore prolong drug half-life [229]. Kidney filtration is an essential function of the renal system. Renal clearance rate associates with several properties, including particle size, shape, and surface charge. For traditional chemical drugs, renal clearance is one of the key points needed in drug delivery [230]. Proper renal clearance helps to minimize toxicity of nanocarrier. These barriers are obstacles for many conventional drug deliveries, diminishing drug efficacy in cancerous sites and indirectly increasing dosage and toxicity for normal tissue.Nanomaterials and BBB The BBB is a highly specialized protection structure that protects the central nervous system from harmful agents and provides essential nutrition. BBB consists of brain capillary endothelial cells, which are arranged to form a “wall.” Due to the blocking function of BBB, current post-surgery chemotherapy methods for brain cancer are mainly intraventricular or intracerebral direct injections, infusion, even implantation. However, these methods aiming at increased permeability might result in risks associated with high toxicity or inadequate drug distribution, that demands for a better solution to deliver anticancer drugs through BBB [231]. In brain tumor treatment, conventional free chemical drugs are hard to reach cancerous sites through intravenous method due to BBB, and nanomaterials are researched to overcome this obstacle. EPR effect, peptide-modified endocytosis and transcytosis, focused ultrasound (FUS) are major approaches currently utilized to help deliver nanomaterials (Fig. 3). Several nanomaterials have been researched for delivery through BBB, including NLCs [232], liposomes, and AuNPs. A glutathione PEGylated liposome loaded with methotrexate (MTX) was tested in rats and the result showed the nanocarrier improves the brain uptake of MTX [233]. AuNPs are vastly researched among these materials. Research concerning glioma and other intracranial cancers are conducted mainly in the mouse model, and the result shows that EPR effect allows gold nanoparticles (AuNPs) of certain size to accumulate in the tumor [234]. To gain better specificity, inorganic NPs such as AuNPs can also be modified with peptides and antibodies on the surface. AuNPs and gold liposomes are used as biocargo for chemical drugs and nucleic acid, and AuNPs are also used in PTT and immune therapy. Ruan et al. fabricated a novel NP AuNPs-A&C-R that were composed of two functional particles, and both particles were peptide modified AuNPs. Peptide attached on the surface helps mediate AuNPs-A&C-R transcytosis across BBB and target receptors on glioblastoma cell surface. This AuNP loaded DOX and showed better chemotherapeutic effect than free DOX treatment [235]. Research indicates that ultrasound can widen BBB tight junction therefore offers a temporary pathway for NPs to get through, and size of AuNPs affects delivery efficiency. This research shows ultrasound might help AuNPs with therapeutic function to penetrate BBB with ultrasound treatment [236] (Fig. 3). Current mouse experiments show that modified AuNPs can help transport chemical drugs, induce lethal autophagy and apoptosis [237] and exert photothermal effect in intracranial cancer PTT [238].Targeting strategies of nanomaterials applied to cancer therapy Targeted therapy aims at specific biological pathways or proteins that function in tumor growth. Molecules related to apoptosis and angiogenesis are also common targets in targeted therapies. Small molecules inhibitors and mAbs are two major tools to be utilized in targeted therapies [14]. Through antigen–antibody conjugation, better specificity can be achieved. Compared with non-targeted therapies, free chemical drugs for example, targeted therapies specifically affect tumor-related molecular targets, while free chemical drugs kill both rapidly dividing normal cells and cancer cells. NPs loaded with targeted therapy drugs or modified with specifically targeted mAbs in the surface gain better efficacy and lower toxicity compared to nanocarriers loaded with anti-tumor chemical drugs (Table 1).The EPR effect is a fundamental mechanism applied in nanocarrier targeting strategy. Passive targeting based on EPR effect involves interactions between the nanoplatform and TME, MPS, and barriers in the human body. It should be noted that EPR also functions in active targeting strategy achieved by conjugating with antibodies, peptides, aptamers and small molecules, and the efficacy of active targeting is affected by MPS, immune system, and other nanocarrier–environment interactions. Both passive targeting and active targeting strategies are used in DDS design. By loading the nanocarrier or modifying the surface with therapeutic ingredients in targeted therapy, the fabricated nanoplatform can be utilized to improve current targeted therapy and achieve better efficacy.Current challenges of nano-DDS designing Three key issues should be considered in anti-cancer nano-DDS designing: enhancement of efficacy, reduction of side effects, and resistance prevention. In many cases, a nano-DDS can solve several problems simultaneously due to instinct mechanism. A SLN synthesized with the material dexamethasone (Dexa)-conjugated lipid is linked with PEG-phosphatidylethanolamine (PEG-PE) and obtains Tf (transferrin)-PEG-PE ligands. As many cancer cells over-express the Tf receptor and use it to obtain certain molecular epitope, Tf is considered the target moiety that binds to the TfR molecular on the HepG2 cells [239]. This kind of surface modification makes it a better delivery vehicle for gene, and the experiment shows that it displays remarkably higher transfection efficiency than both non-modified SLNs/pEGFP and vectors that do not contain Dexa in vitro or in vivo [240]. The increased specificity results in higher drug accumulation in targeted cancer sites than other vital organs, leading to reduced toxicity and drug-related MDR prevention [241].Despite rapidly growing research concerning nanomaterials in cancer treatment, some issues still remain unsolved. Toxicity is still one of the main concerns of nanomaterials. Because of the extremely small size, physiological barriers can be penetrated through, which may pose potential health hazards [242]. Evidence shows that cellular membranes, organelles, and DNA suffer from free radicals caused by NPs [243]. Nanomaterials delivered intracellularly might stimulate an immune response by reacting with cell surface receptors [244, 245]. As referred to above, nanomaterial toxicity relates to many factors and thus, modification to reduce toxicity is essential in the fabrication process.As the primary passive delivery method utilizing nanomaterials, the EPR effect has been closely studied for a long time. However, most designed nanomaterials failed to reach the stage of clinical use. Some researchers tried to re-consider the concept of EPR and explore the real efficacy of this “royal gate” toward cancer treatment. The EPR effect works in rodents differently as in humans [246]. Sindhwani et al. investigated the mechanism by which NPs enter solid tumors. The experiments used four different mouse models, three types of human tumor cells, mathematical simulation and modeling, two imaging techniques, and the results were stunning. The frequency of gaps in tumors did not account for nanoparticle accumulation in tumor. Trans-endothelial pathways were the dominant mechanism of nanoparticle tumor extravasation. Finally, combined evidence from TEM and 3D microscopy showed that there were not enough gaps, which resulted in rare opportunities for cancer nanomedicine to enter tumors passively [247]. These studies indicate that the differences in EPR efficacy in various cells and tissues need further investigations. Studies have been conducted to stratify cancer patients by accumulating NPs through EPR and to find predictive EPR markers [248, 249]. These results indicate that the EPR effect varies in different species and tumors. To better exploit the EPR effect in cancer therapy, more research is needed to explore different patterns and efficiencies of the EPR effect and elucidate the mechanism of nano-carrier transport.Another knotty obstacle of nanomaterial implementation in cancer treatment lies in clinical translation. Although plenty of nanocarrier research for cancer therapy has been conducted (Table 5), most of these researches involve cell and animal models that may not reflect coherent responses in actual human organs. A single model is hard to imitate real reaction in the human body, and previous studies exhibited more consistency of EPR in animals than in human patience [250]. Models of cancer metastasis should also be considered in research as metastasis is common for malignant cancers. The specific solution to these problems is hard to reach; however, innovative modeling methods can be explored to accelerate the process. Biomimetic ‘organ/tumor-on-a-chip’ tools, organoid model systems are possible solutions to imitate in vivo situation of nanocarriers used in cancer patients [251,252,253]. Proper animal models are also recommended in these assessments. Properties of nanomaterials, including size, shape, chemical composition, surface charge, have an enormous influence on nanocarriers' overall efficacy, and adjustment of these properties needs researchers' cooperation in both medicine and material fields. So far, approved nanocarriers used in cancer therapies are mostly liposomes and nanoparticles, and nanocarriers with more complex structures and manufacturing procedures generally face greater difficulties in clinical translation (Table 5). Searching for technology that helps manufacture vast nanomaterials with combined required properties is one important goal in anticancer nanomaterial clinical translation.Table 5 Examples of nanocarriers for anticancer therapyFull size tableProteomics and anti-cancer nanoplatform design When injected into a biological system, nanomaterials are surrounded by serum and cellular proteins, structures formed by these substances are termed protein corona (PC) [261]. Searching for technology that helps manufacture vast nanomaterials with combined required properties is one important goal in anticancer nanomaterial clinical translation. It has been found that since different binding affinities toward NPs are shown by proteins, “hard” corona can form with higher binding affinity proteins, while “soft” corona forms with proteins that bind loosely to nanoparticles. As a result, the most abundant proteins that form a PC first, with time they will be replaced by the proteins with higher affinities. This phenomenon is named as Vroman effect [262]. Various proteomic methods have been used in PC research, especially in quantitative analysis: MS, LC–MS, SDS-PAGE [263], surface plasmon resonance (SPR), isothermal microcalorimetry (ITC). PC affects the interaction of NP with biological environment and therefore, determines whether a NP carrier could be applied in medical use to a degree. Thus, proteomic methods help study NP-protein interaction and achieve a deeper understanding of PC formation.Cancer proteomics analyzes protein quantity in cancer cells and serum, which helps find proteins and surface biomarkers useful in cancer diagnosis and prognosis [264]. Proteomics has also been applied to help understand cancer pathogenesis, elucidate the mechanism of drug resistance, and search for biomarkers for early detection of cancer [265]. In the pathological process, PTMs (post-translational modifications) are important mechanisms related to cancer occurrence, metastasis and reoccurrence, and kinase plays essential roles in these modifications and pathways. Although chemical drugs are the current focus of research, kinase inhibitors and other novel therapeutic agents such as siRNA, mRNA, and gene editing materials cognized through cancer proteomics approaches can be loaded within a nanocarrier to achieve higher drug efficacy. New molecular targets can also be identified by proteomic methods, enriching currently recognized targeting moieties. High throughput proteomics and many novel ways are also enhancing the capability of proteomic methods to identify specific molecules potential for manufacturing anticancer nanocarriers. Conclusions Nanomaterials share similar size but differ in composition, structure, hydrophobicity, magnetism, immunogenicity and other properties. Cancer therapies based on these unique properties have been vastly researched. In general, various surface modification can be achieved on different nanomaterials, and in many cases, conventional anti-tumor chemical drugs can be loaded into different nanocarriers. It is crucial for researchers to be well aware of the characteristics of the selected nanoplatform as well as properties of therapeutic agents. For instance, EVs are biocompatible vesicles with ability to escape the immune surveillance and internalize smoothly with target cells, a possible strategy might be using antibody modified EV to deliver key gene therapy agents to targeted cancer cells. Based on photothermal properties CNTs and metallic materials possess, nanoplatform functions with chemotherapy and PTT can be designed to produce synergistic effect. CNTs have the potential to achieve better anti-tumor efficacy for the feature that they can provide several kinds of therapies at the same time. Both targeted delivery and non-targeted delivery employ nanomaterials as vehicles to transport chemical drugs, peptide/protein molecules, small molecule inhibitors or use the material as immune system stimulant, photothermal medium, chemodynamic medium. Modification of the nanomaterial platform including inner content and external moiety plays an important role in the efficacy, targeting ability, biocompatibility and toxicity of the nanoplatform complex.In this article, we mainly focus on characteristics of common nanomaterials and progress of their application in cancer therapy rather than the chemical synthesis process and drug-loading technique which are also important issues limiting clinical translation of nanomaterials. Targeting therapy and immunotherapy that involve molecules in newly discovered pathways are being massively researched. It is expected in the future, with development in proteomic research on mechanism of cancer genesis, MDR occurrence, more nanomaterial-based targeting therapy and immunotherapy approaches will be explored.Compared to the enormous amount of research, only a few nanomaterial-based drugs are applied in clinical. To improve this situation, more efforts should be taken into toxicity reduction, illumination of EPR and PC mechanism in the human body. It is expected that in the near future, nanoplatforms will be designed to target not only on cancer cells, but also on the TME environment including immune system. Precise targeting methods, TME triggered release strategy, combined therapies, self-assembly nanoplatform are practical approaches to enhance targeting specificity, drug capacity, efficacy, bioavailability; and reduce the toxicity of nanomaterials and loaded drugs toward normal cells. Testing nanomaterials in models that resemble more in vivo environment is also an important issue to be considered. Overall, with the advancement of nanobiotechnology and cancer therapy development, we believe that the breakthrough in clinical translation for treating cancer, a deadly disease, will be achieved, and more nanomaterial-based drugs will benefit cancer patients. Availability of data and materials Not applicable. Abbreviations 5-ALA:5-Aminolevulinic acidAA-PEG:Aminoethylanisamide-polyethylene glycolABCs:ATP-binding cassette transportersADCs:Antibody–drug conjugatesAuNPs:Gold nanoparticlesBBB:Blood–brain barrierBis-MPA:2,2-Bis(hydroxymethyl) propionic acidCA:Citric acidCAR:Chimeric antigen receptorCas:CRISPR-associated proteinsCDP:Cyclodextrin polymerCDs:Carbon dotsCDT:Chemodynamic therapyCe6:Chlorin e6CL:Cationic liposomeCMCS:Carboxymethyl chitosanCNHs:Carbon nanohornsCNTs:Carbon nanotubesCQDs:Carbon quantum dotsCRISPR:Clustered regularly interspaced short palindromic repeatsCSCs:Cancer stem cellsCTLA-4:Cytotoxic T-lymphocyte-associated protein 4DDS:Drug delivery systemDexa:DexamethasoneDOX:DoxorubicinECM:Extracellular matrixEPR:Enhanced permeability and retentionEVs:Extracellular vesiclesexoDOX:Exosomes loaded with DOXFA-SGQDs:Folic acid to sulfur-doped graphene quantum dotsFNPs:Fluorescent polymeric nanoparticlesFUS:Focused ultrasoundGDY:GraphyneGO:Graphene oxideGQDs:Graphene quantum dotsGSH:GlutathioneHA:Hyaluronic acidHAase:HyaluronidaseHER2:Human epidermal growth factor receptor 2HPRG:Histidine–proline-rich glycoproteinHREC:Human retinal endothelial cellsICIs:Immune check point inhibitorsIFN-γ:Cytokine Interferon gammaIFP:High interstitial fluid pressureIHC:ImmunohistochemicalING4:Inhibitor of growth 4IONPs:Iron oxide nanoparticlesITC:Isothermal microcalorimetryL-DOX:Liposomal doxorubicinLUV:Large unilamellar vesiclesmAbs:Monoclonal antibodiesMDR:Multi-drug resistanceMEK/ERK:Mitogen-activated protein kinase kinase/extracellular signal regulated protein kinasemiR-497:MiRNA-497miRNAs:MicroRNAsMLV:Multilamellar vesiclesMMPs:Matrix metalloproteinasesMPA:Mercaptopropionic acidMPS:Mononuclear phagocyte systemMTX:MethotrexateMWCNTs:Multiwalled carbon nanotubesNE:NanoemulsionsNIR:Near-infraredNLCs:Nanostructured lipid carriersNPs:NanoparticlesNSTI:Nanoscience, technology, and industryOS:OsteosarcomaPA:PhotoacousticPAMAM:PolyamidoaminePC:Protein coronaPCL:Poly(ε-caprolactone)PCX:Etoposide and paclitaxelPD-1:Programmed cell death protein 1PD-L1:Programmed cell death ligand 1PDT:Photodynamic therapyPEG:Poly(ethylene glycol)PEG-PE:PEG-phosphatidylethanolamineP-gp:P-glycoproteinPI3K/mTOR:Phosphoinositide 3-kinase/mammalian target of rapamycinPLA:Polylactic acidPLGA:Poly(lactic-co-glycolic acid)PMMA:Polymethyl methacrylatePNPs:Polymeric nanoparticlesPPI:PolypropyleniminePTEN:Phosphatase and tensin homologPTMs:Post-translational modificationsPTT:Photothermal therapyPTX:PaclitaxelrGO:Reduced graphene oxideRGD:Arginine-glycine-asparticRNAi:RNA interferenceROS:Reactive oxygen speciesSDT:Sonodynamic therapySf:SorafenibSi:SiRNAsiRNA:Small interfering RNAsSLNs:Solid lipid nanoparticlesSPION:Superparamagnetic iron oxide nanoparticlesSPR:Surface plasmon resonanceSUV:Small unilamellar vesiclesSWCNTs:Single-walled carbon nanotubesTEA:TriethanolamineTf:TransferrinTLR4/NF-κB:Toll-like receptor 4/nuclear factor kappa BTmab:TrastuzumabTME:Tumor microenvironmentTNF-α:Tumor necrosis factor alphaTPGS:Tocopheryl polyethylene glycol 1000 succinateTRAIL:Tumor necrosis factor-related apoptosis-inducing ligandUV:UltravioletVEGF:Vascular endothelial growth factorReferences 1.Lopez-Soto A, Gonzalez S, Smyth MJ, Galluzzi L. 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Proteomics of ovarian cancer: functional insights and clinical applications. Cancer Metastasis Rev. 2015;34(1):83–96.CAS PubMed PubMed Central Article Google ScholarDownload references Acknowledgements Not applicable. Funding This work was supported by grants from the National Natural Science Foundation of China (81974074 and 81570537), Outstanding Youth Project of Hunan Education Department (19B475). Author information Author notes Zhe Cheng and Maoyu Li have contributed equally to this work.Affiliations Department of Oncology, NHC Key Laboratory of Cancer Proteomics, Laboratory of Structural Biology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, ChinaZhe Cheng, Maoyu Li & Yongheng Chen National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, ChinaMaoyu Li & Yongheng Chen Department of Nucleotide Metabolism and Drug Discovery, The Hormel Institute, University of Minnesota, Austin, MN, 55912, USARaja DeyContributions YC conceived and supervised the project; ZC wrote the paper, ML and RD provided critical suggestions; ZC revised the paper. All authors read and approved the final manuscript.Corresponding author Correspondence to Yongheng Chen. Ethics declarations Ethics approval and consent to participate Not applicable.Consent for publication Not applicable.Competing interests The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Reprints and Permissions About this article 📷Cite this article Cheng, Z., Li, M., Dey, R. et al. Nanomaterials for cancer therapy: current progress and perspectives. J Hematol Oncol 14, 85 (2021). https://doi.org/10.1186/s13045-021-01096-0Download citation Received16 March 2021 Accepted24 May 2021 Publi

Article Nanomaterials for cancer therapy: current progress and perspectives

Maybe you can store them in solution instead of in the solid form.

so one of the limitation of green method is the presence of various reducing agents in the extracts, which is the prime reason for the polydispersity or commonly called as nanoparticle population of various sizes. when you see it in DLS you will observe Polydispersity index or PDI of more than 0.03 and you will see multiple peaks. Furthermore over time they tend to agglomerate, which is going to tell you one of the important thing. The reduction has not completed and the impact of stabalizing agent is not significant to prevent the agglomeration.

If you see that zeta potential of your particle is less than 10, there is a very high probability that your particles will tend to agglomerate. this is becasue of weak steric repulsions in the solution which makes agglomerates.

you can prevent it by adding a surfactant, like tween 20,80 or by doing pegylation.

One more thing which is not a permanent solution is do do sonication of sample prior to SEM. this will break the surface adsorb aggregates and your particle will be uniformly distributed and you will be able to obtain a good SEM, but remember this is not a permanent solution.

Also one of the good thing while performing SEM is to make a very diluted sample, and activate the surface of the glass slide prior to loading your sample that you are going to sputter it later on with gold coating.

I hope this answered some of your queries. Let me know. if there is still something to discuss. we discuss to learn and grow

Although Nancy Ann Watanabe added some interesting information about cytotoxicity of TiO2 NPs of different sizes, I noted that the larger NPs were agglomerates rather than single particles. I suspect that there could be different mechanisms triggered for cell responses base on size alone, but with additional effects that result from the surface morphology of the NPs. Because many important biological entities are les than about 20 nanometers in size (enzymes, antibodies, antigens, etc.), then especially for larger particles (say100-200 nm), I would expect a considerably different surface morphology for a single large particle compare to an agglomeration or aggregation of smaller particles that comprise a larger NP of the same size as the single large particle. These differences of surface morphology are expected to also give rise to differences in surface charge and charge distribution that could result in differences in properties such as binding site affinity and recognition of "foreign" material to be dealt with.