Electroporation of polymeric nanoparticles: an alternative technique for transdermal delivery of insulin
Rachna Rastogi, Sneh Anand and Veena Koul
Introduction:
There have been various techniques developed for delivery of drugs such as insulin into the body. Transdermal delivery of peptides and proteins has drawn interest since the technique allows control of drug input rate relieves patients’ fear of needles. But penetrating the skin barrier, stratum corneum (SC) creates a problem for this technique. Nanosized vesicles made with block copolymers of poly(ε-caprolactone) (PCL) and polyethylene glycol (PEG) are loaded with insulin and delivered to rat abdominal skins (in vivo and in vitro) through application of high voltage and current (electroporation). Studies are then done on the alterations of the skin structure and changes in the blood glucose level to see the effectiveness of the technique.
Methods:
Synthesizing the vesicles:
The copolymer was synthesized in a tri-block orientation: PCL-PEG-PCL. The polymers were then emulsified with 1 mg insulin. Formulation was stirred until nanoparticles are formed. Nile red (NR), a type of dye, was loaded into the vesicles to allow better detection.
Electroporation protocol:
The electroporation process requires a stainless steel pin electrode array clamped to a square wave pulse generator. Effects of voltage, pulse length and number of pulses are determined by insulin flux rate. Depth of penetration by the vesicles is detected by fluorescence microscopy (seeing the NR).
There have been various techniques developed for delivery of drugs such as insulin into the body. Transdermal delivery of peptides and proteins has drawn interest since the technique allows control of drug input rate relieves patients’ fear of needles. But penetrating the skin barrier, stratum corneum (SC) creates a problem for this technique. Nanosized vesicles made with block copolymers of poly(ε-caprolactone) (PCL) and polyethylene glycol (PEG) are loaded with insulin and delivered to rat abdominal skins (in vivo and in vitro) through application of high voltage and current (electroporation). Studies are then done on the alterations of the skin structure and changes in the blood glucose level to see the effectiveness of the technique.
Methods:
Synthesizing the vesicles:
The copolymer was synthesized in a tri-block orientation: PCL-PEG-PCL. The polymers were then emulsified with 1 mg insulin. Formulation was stirred until nanoparticles are formed. Nile red (NR), a type of dye, was loaded into the vesicles to allow better detection.
Electroporation protocol:
The electroporation process requires a stainless steel pin electrode array clamped to a square wave pulse generator. Effects of voltage, pulse length and number of pulses are determined by insulin flux rate. Depth of penetration by the vesicles is detected by fluorescence microscopy (seeing the NR).
In vitro experiment:
Franz diffusion cells, systems composed of receptor and donor cells, are placed on a magnetic stirrer. The insulin-loaded nanovesicles are the donors while the rehydrated rat skin acts as the receptor. Pulses are then introduced to the skin to induce insulin transfer. The insulin concentration was determined by a solid-phase two-site immunoassay (similar to Western Blot). The depth of the nanovesicles is detected using confocal laser scanning microscopy (CLSM).
In vivo experiment:
Male adult Wistar rats are induced with diabetes mellitus and tested with subcutaneous injection of insulin as control. Gel contained with nanovesicles loaded with insulin is then applied on the rats followed by pulsing with pin electrodes. Blood glucose level was monitored and serum insulin level was determined using enzyme-linked-immunosorbent assay (ELISA).
Results and Discussion:
Polymeric vesicles, similar to phospholipid vesicles, can passively permeate through the transdermal membrane, but at a slow rate. Application of electrical current (electroporation) is believed to speed up the transfer rate. Polymeric vesicles were made with insulin entrapment efficiency of around 25-40%.
Franz diffusion cells, systems composed of receptor and donor cells, are placed on a magnetic stirrer. The insulin-loaded nanovesicles are the donors while the rehydrated rat skin acts as the receptor. Pulses are then introduced to the skin to induce insulin transfer. The insulin concentration was determined by a solid-phase two-site immunoassay (similar to Western Blot). The depth of the nanovesicles is detected using confocal laser scanning microscopy (CLSM).
In vivo experiment:
Male adult Wistar rats are induced with diabetes mellitus and tested with subcutaneous injection of insulin as control. Gel contained with nanovesicles loaded with insulin is then applied on the rats followed by pulsing with pin electrodes. Blood glucose level was monitored and serum insulin level was determined using enzyme-linked-immunosorbent assay (ELISA).
Results and Discussion:
Polymeric vesicles, similar to phospholipid vesicles, can passively permeate through the transdermal membrane, but at a slow rate. Application of electrical current (electroporation) is believed to speed up the transfer rate. Polymeric vesicles were made with insulin entrapment efficiency of around 25-40%.
Insulin flux was measured under variable voltage, number of pulses, and pulse length. Passive diffusion of insulin was negligible. Insulin flux changes linearly with increase voltage. The rate of permeation remains elevated several hours after electroporation, suggesting that the prolonged change in permeability may be caused by altered skin structure. A linear increase is also observed with increase in pulse number and pulse length. The max flux of insulin was observed with 10 pulses lasting 15 ms at 100V. 
Figure 3. (A) Effect of pulse voltage, (B) number of pulses, and (C)
pulse length on electroporative insulin flux across rat skin. A good
linear trend was observed with pulse voltage (R2 = 0.9514) and pulse
length (R2 = 0.9937); n = 4 for all experiments.
Histological assessment:
The SC was seen to be detached from the underlying layers in all electroporated samples. Application of electroporation also showed widening of the openings of hair follicles and an increase in ruffling of the epidermis. Recovery of the skin structure at 24 to 48 hours were studied and shown in the figure.
Figure 4. (A) Histological examination of untreated rat abdominal skin. Hair follicles showed separation and increase in pore size as marked.(B) The hair bulbs were also dilated. (C) The stratum corneum (SC) surface showed formation of ‘craters’ or pits with stripping SC layers. Extent of recovery of treated skin at (D) 24 and (E) 48 hours postelectroporation.
Passive treatment vs. electroporation:
Application of electroporation shows about 6.5 times increase in cumulative amount of insulin permeated compared to passive vesicles. The sustained permeation of insulin after the electroporation shows that the vesicles are intact after pulsing.
Figure 5. Top: Comparison of passive and electroporative applicationof loaded vesicles on the permeation of insulin across rat skin.
Bottom figure shows the quantities of insulin retained in the skin
after electroporation of solution and vesicles after 24 hours.
Assessment by CLSM:
The depth of penetration was dependent on the duration of contact with the electrode. A max depth is reach at 60 μm in 24 hours. Vesicles were present throughout the complete depth of the tissue.
Study of Blood Glucose Level (BGL):
Electroporation technique of delivering insulin mimics the blood glucose profile of the IV injection. A slight delay is observed in electroporation due to the slow movement of vesicles across the skin layer. Therefore, compared to IV injection, the effect does not reach the blood circulation instantaneously. Also, electroporation of the nanovesicles has a longer delivery period, giving a possibility for patients to reduce the frequency of administration.
Figure 7. Reduction in blood glucose levels (%) in diabetic Wistar
rats on injectable and electroporative administration of insulin and
insulin-loaded nanoparticles [Control, saline i.v.; insulin (s.c.), subcutaneous
insulin; NP (i.v.), i.v. administration of insulin-loaded
nanoparticles; insulin (electro), electroporative delivery; NP (electro),
electroporation of insulin-loaded nanoparticles]. Inset shows
initial hypoglycemia posttreatment till t = 6 hours. All results
expressed are a mean of 6–8 animals.
The depth of penetration was dependent on the duration of contact with the electrode. A max depth is reach at 60 μm in 24 hours. Vesicles were present throughout the complete depth of the tissue.
Study of Blood Glucose Level (BGL):Electroporation technique of delivering insulin mimics the blood glucose profile of the IV injection. A slight delay is observed in electroporation due to the slow movement of vesicles across the skin layer. Therefore, compared to IV injection, the effect does not reach the blood circulation instantaneously. Also, electroporation of the nanovesicles has a longer delivery period, giving a possibility for patients to reduce the frequency of administration.
Figure 7. Reduction in blood glucose levels (%) in diabetic Wistar
rats on injectable and electroporative administration of insulin and
insulin-loaded nanoparticles [Control, saline i.v.; insulin (s.c.), subcutaneous
insulin; NP (i.v.), i.v. administration of insulin-loaded
nanoparticles; insulin (electro), electroporative delivery; NP (electro),
electroporation of insulin-loaded nanoparticles]. Inset shows
initial hypoglycemia posttreatment till t = 6 hours. All results
expressed are a mean of 6–8 animals.
Conclusion:
Electroporation of nanovesicles can be considered comparable to direct injection as shown by the in vitro and in vivo experiment. In vitro experiment demonstrated that the vesicles penetrate the deeper layers of epidermis while the in vivo experiment shows a prolonged hypoglycemic level up to 36 hours. Further studies need to be done on the effect of vesicle degradation and whether related toxicity may be involved.
Critique:
The paper has showed in depth study on nanoparticle deployment of insulin through electroporation with both an in vivo and in vitro experiment. The in vitro experiment was to demonstrate that the particles are able to penetrate disperse through the layers of the skin and into the bloodstream. The vivo experiment was to show that the blood glucose level can be regulated by the insulin delivered. The results from the tests on the various electroporation parameters seemed slightly suspicious since all three different parameter showed a linear increase, but the paper did mention that other papers have shown similar results. Also, the paper mention that the efficiency of trapping the insulin within the nanoparticle is around 25-40%, yet it does not mention a method to insure that the nanoparticles delivered through electroporation actually contains insulin. Most figures and graphs in the article are easy to interpret except the last one concerning blood glucose level. There were too many components in the graph, which made it hard to comprehend.
Further studies definitely need to done before this technique can be used since the paper itself stated that mouse skins are not good test subjects since it differs greatly from human skins. Also, as mentioned in the conclusion, effects of the byproducts from the degradation of the nanopaticles are still unknown.
Electroporation of nanovesicles can be considered comparable to direct injection as shown by the in vitro and in vivo experiment. In vitro experiment demonstrated that the vesicles penetrate the deeper layers of epidermis while the in vivo experiment shows a prolonged hypoglycemic level up to 36 hours. Further studies need to be done on the effect of vesicle degradation and whether related toxicity may be involved.
Critique:
The paper has showed in depth study on nanoparticle deployment of insulin through electroporation with both an in vivo and in vitro experiment. The in vitro experiment was to demonstrate that the particles are able to penetrate disperse through the layers of the skin and into the bloodstream. The vivo experiment was to show that the blood glucose level can be regulated by the insulin delivered. The results from the tests on the various electroporation parameters seemed slightly suspicious since all three different parameter showed a linear increase, but the paper did mention that other papers have shown similar results. Also, the paper mention that the efficiency of trapping the insulin within the nanoparticle is around 25-40%, yet it does not mention a method to insure that the nanoparticles delivered through electroporation actually contains insulin. Most figures and graphs in the article are easy to interpret except the last one concerning blood glucose level. There were too many components in the graph, which made it hard to comprehend.
Further studies definitely need to done before this technique can be used since the paper itself stated that mouse skins are not good test subjects since it differs greatly from human skins. Also, as mentioned in the conclusion, effects of the byproducts from the degradation of the nanopaticles are still unknown.
8 comments:
I thought that the purpose of this new drug delivery method was to provide an alternative to insulin injection: it seems that with the electroporation method you are still using needles in the skin in order to create an electric field. Also, isn't the pulsing with electrodes possibly harmful to the skin itself? I don't know if this paper has effectively provided a less painful or dangerous method of insulin injection, although the fact that the insulin has a more sustained release is a definite benefit since you wouldn't need to do as many injections. I agree with Chia-Hung that 25-40% efficiency of trapping insulin within the nanoparticles is too low: if this method were to be used an increased efficiency percentage is definitely needed.
I agree with Danielle in that this is hardly a "better" alternative to using needles in patients. I don't like how this paper does not address the pain aspects with regards to electrophoresis, especially when it's purpose is to address the pain of having to use needles. Another concern that comes to mind is the electroporation of random contaminants that might occur from a patient using this method? Shouldn't the paper also address the degradation time of PCL/PEG in the body? I'm pretty sure it wouldn't degrade within 2-3 days unless it was amorphous or something, which probably isnt the case because of its regular triblock configuration.
Either this could only possibly function as a painful slow release method of insulin as an alternative to frequent injections of insulin or it wouldn't work at all. I also think this would damage the skin over time or might leave long term effects in this nature. This really doesn't seem like a good device to pursue.
I agree with the previous posts that this method, although shown by the paper to increase insulin delivery efficiency, seems very painful and harmful to the skin if performed frequently on a long term basis. However, the nanoparticles seems like a promising idea because of their ability for sustained release, if there was some other way to implant them into the body that was less damaging.
I agree with the previous comments. This approach will obviously be very expensive and will only be applicable if there is minimal or no patient pain.
Carlos, in regards to the triblock polymer, I found a paper discussing a PCL-PEG-PCL polymer lasting at least 45 days in vivo (http://onlinelibrary.wiley.com/doi/10.1002/app.31654/pdf). However, since PCL-PEG-PCL nanoparticles have a high surface area to volume ratio, their degradation is likely to be less than this time frame. As you mentioned, this degradation time must be accessed.
Instead of using PCL-PEG-PCL nanoparticles, I think a coblock PGA-PLL polymer should be used. Not only does this polymer have a lower degradation time (2 weeks), but it is also FDA approved for many drug delivery systems. If this polymer were used, the toxicity of the byproducts wouldn't need to be accessed saving time and money.
Finally, to increase the insulin within the nanoparticle, a double emulsion synthesis technique should be used rather than a single emulsion.
Although the paper mentioned that the needle were blunt end needles, i am not sure whether it will lower the fear factor of patients. If the goal is to avoid pain to reduce the fear from patients, then this paper should perform experiments showing how much "pain" this technique is relative to normal needle injection. Also, from the electrical setup, it seems to need large voltage to generate enough E field to inject the drugs, which in turn may cause substantial pain. I think the authors need to justify a better reason for developing this sort of setup.
As many have mentioned before, it seems that this technique, as it stands now, is not a better alternative to insulin injections. If anything, optimization of this procedure and alteration perhaps of the nanoparticle compononents itself is required. I would have like to have the authors mention at what voltage and duration of exposure becomes harmful and induces injury to their subjects. I imagine, as someone mentioned before, that since mouse and human skin vary substantially, it may not be informative for human subjects. However, at least it gives the reader an estimation of how much physical damage is done during this process. Since insulin injections are taken daily for many, repeated exposure to this process may cause chronic damage that has not been addressed in this study.
Finally, it would have been nice if the authors did some preliminary studies to see the associated toxic effects of this procedure and nanoparticle delivery and the associated immune respnse. If the immune response is too great, I would imagine that some of the insulin delivery efficiency would be reduced.
@ Danielle: The electrode can be somewhat harmful to the skin, which leads to scarring. Also, sometimes the pores take much longer for it to reseal, which leads to Carlos’s question. I do think that the procedure needs to be performed in a clean environment, or the bacteria and other contaminants can enter through the enlarged pores.
I personally believe that in vivo electroporation is not a viable solution right now as a replacement for drug delivery (at least not for insulin anyway). The cost of performing the technique much exceeds that normal syringe injection. Also, improvement in insulin delivery (insulin pumps, insulin pens) are continually evolving to be much more user friendly. The delivery technique, though, may be used to deliver particles that are much smaller and harder to introduce into the body, but efficiency should be improved before using this as a treatment of any type.
I believe the previous comments brought up very good points. The efficiency mentioned in the paper for drug delivery is rather low, and the use of needles still makes it similar to insulin injections. There are many issues they need to address to propose this as an alternative for delivery of insulin
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