The shock might be due to a drop in blood pressure, we have also observed the same thing. To prevent this from happening you should inject the sample very very slowly. Anyway, I think you're injecting a large number of particles, we have observed signs of systemic toxicity above 15 mg / kg, so maybe you should try injecting a diluted sample. Hope this helps. Regarding the biodistribution of nanoparticles, size is very important. You said you are using 50 nm-sized nanoparticles, but are you referring to hydrodynamic size or core size?

Do you mean that the animal received 50 mg/kg?

As we don't know the age or weight of the animals, we can't really deduce how concnetrated/viscous the injection solution was.

Is the 50 mg figure based on particle mass, or just the titanium?

What was the injection rate? Manual delivery, or programmable syringe system?

If you're limited to a 200 ul volume (which is quite significant from the perspective of a mouse), can you deliver it over a course of several minutes?

I presume the animals were not anaesthetised?

Have you tested the likelihood that these particles will aggregate in blood? If they don't significantly aggregate, then I don't see why reducing the concentration would affect RES/MPS clearance.

What is the size of your PEG chain? Is it attached to the particle by both ends of the chain, or by one end only? Are any other surface molecules exposed, or is PEG the only surface feature?

What is the size of these particles when in blood?

Where is the tumour?

(edited to correct spelling mistake)

I had a similar experience. Everything was fine when I used 0.15 mg of nanoparticles/0.1 ml suspension and intravenously administer 0.1 ml/100g animals...

Very important for safety is the complete mono dispersion of the nano particles

Most nanoparticles are taken up by the RES, this is dependent on size being that the larger the particle the faster it is taken up. I have looked at iron oxide particles with the same coating but at different sizes and the small the size (

Lucky, monodisperse your solution. Try less viscous solution or slowly administration. Possible explanation is the higher viscosity of the bloody with a drop in blood return to the heart.

I'm agree with Raquel and Stuart ! 200 µl is quite high for a mouse and viscosity could induce high trouble ! dilution and a slow very slow inoculation with a programmable syringe

Basically two explanations come to my mind. One is the temperature of the solution. If it's too cold , the mice might undergo a short period of arrhytmia or even arrest. 200ul is about 20% of the total blood volume of a mouse Did you test injectin over a longer period of time e.g. 3 min? Did you warm the solution to body temperature?

The second has been mentioned already. Aggregation of particles may increase the size, which would lead to capillary embolism in the lungs, liver and abdominal organs first. Pegylation may be a solution.

From a physiological standpoint th high viscosity is also an acute event for cardiac function. Again injection over a longer period of time may help to dilute your solution in the blood stream.

I think, if you inject the same solution without nanoparticles and evaluate the differences in their reaction. It will exlude an impact of solution and injection pain.

You can use the magnetic functionalized nanoparticle (coupled with biological substances). For nanoparticle location you can use an external magnetic field.

I'm with Ospan and many of the others, try injecting the same solution in the absence of nanoparticles under the same conditions.....and yes, 200 ul is a lot for a mouse....also a slower injection rate might be in order, as already mentioned in this thread.

Hi,

I do agree with everybody else about trying to slow down your injection rate and checking on your aggregation state. One other possibility that occurs to me (from personal previous -bad- experience) is that your suspension may be perceived as hyper-osmotic to the red blood cells. Meaning, you have salts (PBS) and nanoparticles that can account for creating a higher osmotic pressure outside red blood cells than inside. Red blood cells can't cope with that by equilibrating salts. So they will release water molecules from the inside to the outside, in an attempt to 'dilute' their external environment. Consequently, they shrink, so they don't function normally, i.e. they don't transport gases correctly anymore. This is a reversible phenomenon, the situation reverses back to normal after a few minutes. In the meanwhile, it can produce a shock to the mice, even provoking paralysis. To check on that, you have to place your injected suspension on one side of a semi-permeable membrane (a dialysis bag, for example) and put an iso-osmotic solution on the other side (NaCl 0.9% will do just well). If the bag volume remains the same, you injected solution is iso-osmotic (perfect case). If the bag swells, bad news, your suspension is hypo-osmotic and needs to be strengthen by salts. If the bag shrinks, your suspension is hyper-osmotic and needs (again...) to be diluted or to be injected very, very slowly. The more shrinking, the slower the suspension must be injected.

Hope this was helpful. Good luck with your work !

Oh...forgot ...if you do not coat the particles with PEG of the correct chain lenght, the RES system will quickly remove them from the ciculation and few particles reach the tumor (alternative carboxymethyl-Inulin coating and many others..see the reviews)...also targeting tumor surface receptors might help. Michaela's suggestion is another option for concentrating the particles in a particular body area, titanniumdioxide polymorphs, anatase, brookite, and rutile are all diamagnetic solids, , but you might need to dope them for maximum effect ( C or Co).

Some papers:

Heterogeneity in nanoparticles influences biodistribution and targeting.

Adjei IM, Peetla C, Labhasetwar V.

Nanomedicine (Lond). 2013 Jun 26

Nanoparticles-mediated drug delivery approaches for cancer targeting: a review.

Sultana S, Khan MR, Kumar M, Kumar S, Ali M.

J Drug Target. 2013 Feb;21(2):107-25. doi: 10.3109/1061186X.2012.712130

In vivo biodistribution of nanoparticles.

Almeida JP, Chen AL, Foster A, Drezek R.

Nanomedicine (Lond). 2011 Jul;6(5):815-35

The rise and rise of stealth nanocarriers for cancer therapy: passive versus active targeting.

Huynh NT, Roger E, Lautram N, Benoît JP, Passirani C.

Nanomedicine (Lond). 2010 Nov;5(9):1415-33.

Factors that control the circulation time of nanoparticles in blood: challenges, solutions and future prospects.

Yoo JW, Chambers E, Mitragotri S.

Curr Pharm Des. 2010 Jul;16(21):2298-307

The first place any IV injection goes is the lungs, any particulate matter risks causing an embolism in the capillaries, you are also diluting the venous blood, which hits the lungs and can cause poor oxygen transfer. 200ul is a large volume for a mouse (particularly IV), but the viscosity of the solution is likely reducing mixing and the particulates are likely adding to the problem. you may want to think about a slow infusion, or a series of injections to help reduce the particulate load and the viscocity.

Hi, enjoying all the answers above, I would like to add the following concerning my research field. If you inject IV in the tail vein, with such amount of nano particles, I can imagine you damage or at least slow down blood flow in the pulmonary circulation, as this is the first, and only, capillary vessel bed your viscous solution has to pass, before entering the systemic circulation. This results in a ventilation/perfusion mismatch, which could cause the shock you describe in the animals. In that case, prolonging the injection time would less beneficial compared of dosing 5 times 1/5 of your administration. Good luck, and please share your thoughts on this.

I agree with all above answers. Another important consideration is making sure you have very low endotoxin levels for your nanoparticle suspensions you are injecting.

Looks like respiratory distress to me.

Quote: "In all species, injection of compounds that contain particulate material or are of low pH that precipitate when mixed with blood can result in vascular occlusion, emboli, and thrombosis of local and distant capillary beds such as those found in the lungs" (Administration of Substances to Laboratory Animals: Routes of Administration and Factors to Consider, Patricia V Turner et al., 2011, JAALAS).

Why would you use something with already proven toxicity after parental administration? (J Appl Toxicol. 2009, In vivo acute toxicity of titanium dioxide nanoparticles to mice after intraperitioneal injection. Chen J, Dong X, Zhao J, Tang G. )

Sorry, parenteral of course.

Thank you for all the answers, really appreciate it.

Dr.Mejias, my nanoparticle is actually a core-shell nanoparticle with heavy metals in the core, That's why the total weight is high. TiO2 is on the shell. We chose 50 mg/kg based on other similar literature. I agree with you on administering the drug/ nanoparticle suspension into the animal very slowly. But what do you think will be the right duration. I do not anaesthetise the animal during IV injection. Not sure if the animal will move if I inject slowly over a period of time.

By the way, the size (50 nm) is the size recorded by TEM, not the hydrodynamic size. Thank you so much for the answer.

Dr Jenkins,

The age of the animal is about 6-8 weeks and the average weight is about 25g

50 mg is based on the weight of the whole particle. As I mentioned in the reply above, my nanoparticle is actually a core-shell nanoparticle with heavy metals in the core, That's why the total weight is high. TiO2 is on the shell.

The administration was done manually, and was done quite fast, which could be one of the reason as the others pointed out. Yes, I will try to inject slowly and see if it makes a difference. But could you tell me how slowly this must be done. The animals were not anaesthetised.

I have not tested the aggregation in blood. I will try to do that. Do you have any papers on how to do this?

However, I have tested the aggregation of this particles before and after modification with PEG in different media, like water, PBS, culture media with and without serum.The unmodified particles did aggregate in different physiological solution, that is the reason why we had to surface modify the particles. The PEG modified particles had reduced aggregation in the above solutions, however the hydrodynamic sizes were always 6 times higher (300 nm) compared to 50 nm (TEM). But it remained stable over time and did not grow larger.

Size of the PEG chain is 3000Da, attached to the particle at one end only. We have also attached some antibodies to the PEG for targeted delivery. But I observed this behaviour in animals injected with PEG alone particles too.

I have not checked the size of the particles in blood. Can I do DLS measurement to check that?

Tumour is a subcutaneous xenograft tumour on the flanks of the animal.

Dear Dr.Kalber,

Thanks for you answer. Yes, the particles did aggregate over time in PBS, and that is the reason why we did surface modification with PEG. After modification the hydrodynamic sizes stabilised. However, the hydrodynamic sizes were 6 time higher (about 300nm) than the TEM size of 50 nm. Is this normal?

In-vitro work showed the a concentration greater that 250 ug/ml could reduce the viability of the cells to less than 80%. But how can this be translated to in-vivo?

Dear Dr.Roullin,

Thank you for your answer and the wonderful explanation on how to check whether my nanoparticle solution is hyper-tonic. It seems to be a very simple yet important experiment to be performed to check the hemocompatibility of my nanoparticle suspension. Will definitely try that! Thanks

Dear Dr. Kennedy,

Thank you for the answer and the good explanation on what might likely happen to the nanoparticles once in the blood. Yes, I agree with you that the particulates in the suspension may affect the lung and cause capillary embolism and reduced oxygen transfer. So do you think, this will be the reason for the state of shock that I observed? The animals, return to its normal self with in few mins, could it be because the damage is reversible or is because it adapts to the pain or discomfort caused?

Dear Dr.Raaf,

First of all thanks for the answer. Yes, I now understand that it could be due to the particles directly hitting the lung causing a ventillation/perfusion mismatch. Since I am not experience in in-vivo work, and not familiar with the physiology of mouse, I do not know what that means. But I will try to read up, and understand what this mismatch is all about.Thank you once again!

Naturally, the viscocity should be quite high wit the heavy nanoparticles and the shear pain itself can cause the kind of shock described.

Dear Lucky, If you need any assistance (regarding the lungs), please let me know. I am happy to help you.

There is also possibility that your NPs are too big and e.g. have difficulties with coming through the smallest blood vessels.

Because of the viscosity try to treat this injection as the vitamin injection given to humans - do it veeeery slowly. I would recomend even using a syrringe pump.

The viscocity of you solution appears to me the main problem of the brief state of shock and this is certainly due to the diameter of the nanoparticles. Are they functionalized? This can also increase the viscosity.

Anyway, 50nm diameter is quite large, but should not be a problem to travel into the vessel (~5-10 micrometer of diameter). It should be easier to reach the tumour with smaller nanoparticles or as mentioned above. In addition,superparamagnetic nanoparticles are also studied as a solution for guiding them into the tumour.

In fact, the main problem your are facing here about the difficulty to reach the tumour, is the outward convecting interstial fluid flow coming from the internal vascularity of the tumour. It depends on the tumour size, but I guess you are working with developed tumour. So the internal vascularity will decrease the drug diffusion or nanoparticles diffusion into the centre of the tumour. However, this fact is also used as an advantage if you are able to load the drug/nanoparticles inside the tumour as the retention times will be higher than in normal tissues. Hope this will help you to solve your problem.

Good luck in your experiment,

in response to your follow up question. If the animals are recovering then you are unlikely to be causing major lasting damage, full embolism is most often fatal. it may be that you will have pockets of continued embolism and you may want to look at the lungs when you euthanize the animals to make sure that this is not the case (gross inspection should reveal white spots in otherwise pink lungs). Depending on your endpoint, you may want to think carefully about how your vehicle controls for your treatment. you may want to use some form of control nanoparticle rather than plain vehicle.

I agree on the idea of slow down on your injection and check on your formulation. It may be that your formulation has caused hemodynamic instability or vasovagal response.

The volume administred (200 uL) is not very high for mice? In "A Good Practice Guide to the Administration of Substances and Removal of Blood, Including Routes and Volumes" recommends using 5 mL/kg in IV bolus administration for mice. For a mice of 25 g weight, use maximum 125 uL.

Reference: JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 21, 15–23 (2001).

Dear Esteemed Colleague,

I hope this message finds you well. Your observation of animals entering a state of shock following intravenous (IV) injections of nanoparticle suspension raises important considerations regarding the biocompatibility and physiological responses to nanomaterials. Such reactions can be alarming and underscore the necessity for careful evaluation of nanoparticle formulations intended for biomedical applications. Below, I discuss several potential mechanisms that could contribute to this adverse reaction:

1. Particle Size and Aggregation:Nanoparticles with certain sizes or tendencies to aggregate once in the bloodstream may lead to capillary blockage, disrupting blood flow and oxygen delivery to vital organs. This can trigger hypoxia and contribute to shock-like symptoms.

2. Surface Properties:The surface charge, hydrophobicity, and the presence of specific surface coatings on nanoparticles can significantly influence their interaction with blood components, potentially leading to activation of the complement system. This activation can result in an acute immune response, manifesting as symptoms of shock.

3. Material Composition:Certain materials used to create nanoparticles can be inherently toxic or elicit a strong immune response. The composition of the nanoparticles might lead to cytotoxicity, systemic inflammation, or anaphylactic reactions, all of which can precipitate shock.

4. Dose and Administration Rate:The dosage and the rate at which nanoparticles are introduced into the bloodstream can impact the body's response. Rapid administration or high doses may overwhelm the body's clearance mechanisms, leading to accumulation and systemic toxicity.

5. Endotoxin Contamination:Endotoxin contamination in nanoparticle suspensions, possibly arising during synthesis or from biological components used in formulation, can induce shock. Endotoxins are potent stimulators of the immune response and can lead to septic shock if introduced intravenously.

Mitigation Strategies:

• Thorough Characterization: Comprehensive physicochemical characterization of nanoparticles prior to in vivo studies, including size, charge, aggregation state, and surface properties.
• Biocompatibility Testing: In vitro and in vivo biocompatibility assessments to identify potential cytotoxicity, immunogenicity, and hemocompatibility issues.
• Dose Optimization: Careful determination of the optimal dosing and administration rates through preliminary studies.
• Endotoxin Testing: Routine testing for endotoxin levels in nanoparticle formulations intended for IV administration to ensure safety.
• Gradual Dose Escalation: Implementing a gradual dose escalation strategy during in vivo studies to monitor for adverse reactions and determine safe dosage thresholds.

In conclusion, the reaction you've observed underscores the importance of comprehensive preclinical evaluation of nanoparticle formulations for safety and biocompatibility. Understanding and mitigating the underlying causes of such adverse reactions is crucial for the successful application of nanotechnology in medicine.

Should you require further assistance or wish to discuss this matter in more detail, please do not hesitate to contact me.

Best regards,

This list of protocols might help us better address the issue.