2013 年 36 巻 8 号 p. 1283-1291
Percutaneous immune method is becoming an attractive alternative for DNA vaccine as a lot of antigen presenting cells are existed in the viable epidermis. However, due to the barrier function of stratum corneum, it would be hard for DNA vaccine to reach the viable epidermis of the skin. In order to deliver the DNA vaccine successfully cross the stratum corneum, pentagram silicon microneedle array was prepared in this study, and fluorescently labeled nanoparticle was taken as the model to observe the situation inside the skin processed by microneedle. Via microneedle nanoparticles could enter the skin through the micro-channel (diameter about 20–30 µm) and its amount is greatly larger than that enter though the hair follicle of intact skin. A new type of gene vector Pluronic P123-modified polyethyleneimine (P123-PEI) was synthesized by high molecular weight polyethylenimine and Pluronic P123 with the molar ratio of 1 : 1 to take the advantage of P123-PEI as low cytotoxicity and high transfection efficiency. Mice were immunized percutaneously with Hepatitis B DNA vaccine/P123-PEI nano-complexes by microneedle. The humoral and cellular immunity generated in percutaneously immunized mice through microneedle array by Hepatitis B DNA vaccine/P123-PEI nano-complex was significantly higher than that of DNA vaccine intramuscular administration.
DNA vaccine that the eukaryon expression plasmid encoded by antigen gene was inoculated in vivo and can express corresponding antigen to stimulate the organism to generate immune response to the antigen and protective immunity was the third generation vaccine on the basis of gene therapy and transgenic technology, which marked a vaccine revolution and has a wide application prospect.1) However, DNA vaccines had its own disadvantage. Numerous experiments, particularly big animal experiments and human clinical trials, demonstrated that currently DNA vaccine had the disadvantages such as large dose, low bioavailability, great variation in immunity effect among individuals and low level of humoral immunity induced etc. The disadvantage of DNA vaccine enormously affected the research progress and the wide clinical application.2) According to in vivo immune mechanism of DNA vaccine, antigen presenting cells (APCs) like phagocytes and dendritic cells was playing an important role in the process of transfection, expression and presentation of DNA vaccine.3,4) How to improve the cell transfection efficiency of DNA vaccine, particularly the transfection efficiency of APCs, becomes one of the key approaches for improvement of immunity effect of DNA vaccines. Currently, the dosage forms of DNA vaccine are mainly water solution or lyophilized powder injection and intramuscular injection is the most common route of immune administration. DNA vaccine is usually consisted of 2–10 kbp plasmid DNA and his molecular weight reached million with a strong hydrophilicity and low oil–water partition coefficients. After intramuscular administration, it would be hard for DNA vaccine to penetrate cell membrane by means of diffusion model and transfer to histocyte like muscular cell. Therefore antigen presenting cells such as phagocytes and dendritic cell transfer efficiency is particularly low and only few amount of DNA vaccine can be successfully transferred to APCs, which induce low transfection and expressing, lower generating antigen and presenting to generate immunity efficiency. Most of the DNA vaccines are degraded before transferring to cells, which is the reason for the low antigen expression and presenting level and the unsatisfactory immunity effect. The plasmid DNA that failed to go into cells would be easily to be transferred to other organs or tissues outside the injection parts by diffusion, which also cause potential safety issues.5,6) Therefore, to develop a new drug delivery system, which aims to strengthen immunity effect of DNA vaccine, enhance the safety and reduce dose and cost of medication, is the urgent request in the current study on DNA vaccine.
As well known there are three layers of tissue in human skin: stratum corneum, viable epidermis and dermis. The outermost stratum corneum, which is composed of compact keratinocyte with a thickness of 10–15 µm, is the primary obstacle for drug delivery. Below stratum corneum it is viable epidermis with a thickness of 50–100 µm, which contains active cells and small amount of nervous tissues. In the viable epidermis, dendritic cells (DCs). Langerhans cells (LCs, 500–1000 cells mm−2) are existed. Although the Langerhans cells only account for 2% of the total amount of viable epidermis cells, they cover nearly over 20% of body surface area through their horizontal orientation and long protrusions which form a meshwork that allows them to uptake antigens that they encounter, and LCs belong to strong antigen presenting cells.7) When DNA vaccines enter the viable epidermis, where LCs are located, and transfect LCs, express antigen and present antigen to T lymphocyte, immune response is activiated. At the same time, LCs secret major cytokines which would be needed in the process of T cell response and participate in several links of immune responses.8) The immune response caused by LCs is 1000 times higher than that caused by keratinocyte, fibroblast or myocyte.9) Therefore, the viable epidermis is considered as the excellent location for vaccine administration.10,11) Due to the barrier of stratum corneum, DNA vaccine is unable to penetrate the stratum corneum and reach the viable epidermis under physiological condition if conventional chemical penetration enhancer is used. Currently, a few methods of physical penetration enhancer, such as electroporation,12) radio frequency ablation13) and gene gun,14) are used for DNA vaccine percutaneous administration. Recently, the advantage of microneedles as a new drug transdermal delivery system is realized and recognized gradually. Microneedle made by micro electronic mechanical system (MEMS) is referred to a needle-shape complicated structure with a micrometer size and a length of over 100 µm, and the material can be silicon, polymer, metal, etc. Microneedle can provide channels for drug delivery by simply penetrating the stratum corneum without touching the nerve of deep tissue, so it would be no any pain or stimulation. Different depth of the skin can be located according to the length of microneedle.15) Microneedle has some superior characteristics compared with conventional methods such as veracity, analgesia, efficiency and convenience. According to the articles reported, the usage of microneedle in mouse skin can pierce the stratum corneum and thus form the channel for drug delivery. Applying DNA vaccine on corresponding skin, the marked improvement of immune response can be observed.16)
To further enhance the cell transfection efficiency of DNA vaccine and strengthen the immune response, a stable polyplexes was formed by DNA vaccine integrated with cationic polymer by means of electrostatic adsorption to protect DNA vaccine from being degraded by nuclease and to enhance the capacity of the transfection cells in our study. This complex can reach the viable epidermis through the micro-channel on skin formed by microneedle and its positively charge can help DNA vaccine express efficiently by mediated gene taken inside the cells.17) The cationic polymer selected in this study was Pluronic P123-modified polyethyleneimine (P123-PEI). As the gene vector of cationic polymer PEI was studied most comprehensively with advantages of low price, large carrying capacity, no immunogenicity and high transfection efficiency. But it also held certain shortages especially its high cytotoxicity. Meanwhile the particle size and dispersion stability of the complexes formed by PEI and DNA were quite sensitive to buffer system of body fluid, which caused high toxicity, low transfection efficiency, poor pharmacokinetics and distribution in vivo.18) To overcome the disadvantage, this study used Pluronic P123 to modify PEI. Pluronic P123 is a triblock copolymer with hydrophilic polyethylene oxide (PEO) chain and hydrophobic polypropylene oxide (PPO) chain. The modified PEI was expected to improve the colloid stability of complexes and reduce cytotoxicity by means of P123 hydrophilic branched chain as well as keeping the affinity of complexes for cell membrane to get better transfection efficiency.
The strategy of this study was to provide micro-channel to penetrate the stratum corneum and reach the viable epidermis by means of microneedle and then deliver the complexes by DNA vaccine and cationic polymer P123-PEI through the channel. By this manner, efficient transfection, improvement on the uptake of APCs to induce immune response as well as enhance the immunity effect of DNA vaccine could be well realized.
Hepatitis B DNA vaccine (pVAX-S) was kindly supplied by the Department of Medical Genetics of Second Military Medical University. Pluronic P123 (molecular weight (Mw)=5780) was kindly supplied by BASF Company (Ludwigshafen, Germany). Branched Polyethyleneimine (Mw=25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.); 4-methoxytriphenylmethyl chloride and bis(trichloromethyl)carbonate were purchased from Darui Finechem Co., Ltd. (Shanghai, China). N-Hydroxysuccinimide was purchased from Medpep Co., Ltd. (Shanghai, China). Anhydrous triethylamine, anhydrous toluene and anhydrous dichloromethane were purchased from Sino.pharm Chemical Reagent Co., Ltd. (Shanghai, China). Fetal calf serum, DMEM culture solution, PBS buffer solution, penicillin and streptomycin were purchased from Gibco Co. (Shanghai, China). Fluorescence enzyme analytical system and pGL3-Control were purchased from Promega Co. (Madison, WI, U.S.A.). Recombinant hepatitis B surface antigens (HBsAg) protein was purchased from Hissen Bio-pharm. Co., Ltd. (Dalian, China); interferon-gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) determination kit for mouse were purchased from Bender MedSystems Co. (Vienna, Austra); Anti-HBsAg antibody estimation kit was purchased from Kehua Bio-engineering Co., Ltd. (Shanghai, China). Hela cells were purchased from Institute of Biochemistry and Cell Biology of Shanghai Institutes for Biological Sciences, CAS (Shanghai, China).
C57BL/6 mice, female, aged from 6–8 weeks, purchased from Shanghai Laboratory Animal Center, CAS (Shanghai, China), were acclimated at 25°C and 55% of humidity under natural light/dark conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Changhai Hospital, Second Military Medical University (Shanghai, China).
Synthesis and Representation of Cationic Polymer P123-PEIFirst, Pluronic P123 (2.90g, 0.5 mmol) was dried in vacuum at 50°C for 24 h and then dissolved in 40 mL of anhydrous pyridine. Then 4-methoxytriphenylmethyl chloride with equivalent molar weight was added and the mixture was reacted for 4 h at 25°C with nitrogen protection. Thin-layer chromatography was used to test whether the reaction was complete. The solvents were removed under reduced pressure and the precipitate was re-dissolved in 10 mL of dichloromethane, and purified on a silica gel column (230–400 mesh) by using a mixture of methylene chloride and methanol with increasing polarity. After evaporation of the eluent, slightly yellow product was obtained.
The product (0.5 mmol) obtained was dissolved in the mixed solvent of toluene–dichloromethane (3 : 1, 40 mL) and then bis(trichloromethyl)carbonate (0.149 g, 0.5 mmol) was added and the mixture reacted with magnetic stirring for 24 h. The solvents were removed under reduced pressure. The residual was re-dissolved in the mixed solvent of toluene–dichloromethane (2 : 1, 30 mL); N-hydroxysuccinimide (0.060 g, 0.5 mmol) was added and then anhydrous triethylamine (0.071 mL, 0.5 mmol) was added drop by drop; the mixture was reacted with magnetic stirring for 4 h under the protection of Nitrogen. The reaction solution was filtered and evaporated to dryness in vacuum. The product was purified by a silica gel column.
PEI (0.25 g, 0.01 mmol) was dissolved in 10 mL of 10% aqueous ethanol and hydroxyl activated P123 (0.01 mmol) with equivalent molar weight was added. The mixture was reacted with magnetic stirring for 24 h at room temperature and then concentrated hydrochloric acid was added to adjust pH value to 2–3. This reaction was carried out for 30 min and the reaction solution was adjusted to neutral. Sephadex G-75 gel column was used to separate the unconnected PEI with 10% ethanol water as the eluent. The collected product of eluent and unreacted PEI were confirmed by color reaction with picrylsulfonic acid. The eluent, after dialysis of Amicon Ultra-4 membrane tubes (Millipore, U.S.A.) with molecular weight cutoff of 10 kDa against water (twice replaced) for 12 h, were freeze-dried. The white solid was obtained. The composition of P123-PEI was analysised by 1H-NMR (300 MHz, Varian, U.S.A.). Gel permeation chromatography (LC-10A, Shimadzu, Japan) was used to determine the polydispersity of P123-PEI.
Investigation on P123-PEI as Gene VectorCertain amounts of plasmid DNA and PEI or P123-PEI were dissolved separately in 5% glucose solution at pH 7.5. Mixed them with vortexing and incubate at room temperature for 30 min. Then the complexes with different N/P ratio were prepared. One percent (w/v) agarose gel electrophoresis was got to analyze (100 V, 60 min) the ability of PEI or P123-PEI in composite wrapping plasmid DNA at different N/P ratio (N/P=0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10).
Based on different N/P ratio (N/P=6, 9, 12, 24, 48) , the solution (250 µL of 5% glucose solution) with a certain amount of PEI or P123-PEI was mixed with the solution (250 µL of 5% glucose solution) with 20 µg DNA by vortexing and incubated at room temperature for 30 min. The particle size and zeta potential of the complexes were measured by an electrophoretic light scattering spectrophotometer (Zeta Potential/Particle Sizer, 380ZLS, Nicomp™, U.S.A.). The transfection efficiency of the complexes was measured in vitro and the process was briefed as follows. Hela cells were seeded into the 24-well plate with the density 1×105 cells/well, and incubated for 18–24 h. The plasmid of 3 µg pGL3 was formed complex with PEI or P123-PEI at different N/P ratio (N/P=3, 6, 12, 24) and incubated for 30 min at room temperature. The complexes were dispersed in serum-free DMEM and the added to the 24-well plate, and incubated for 5 h at 37°C under a 5% CO2 atmosphere. The media were replaced by fresh growth medium with serum and the cells were incubated for 48 h. The Luciferase assay was carried out by following manufacturer’s instructions (Promega). The amount of cell protein in 20 µL cell extracting solution was determined with Micro-BCA Protein determination kit. The transfection efficiency was expressed with relative light units (RLUs)/mg. All transfection experiments were repeated for three times. And 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of the complexes.
Preparation of Microneedle and Its Role of Penetration EnhancementStar-shaped silicon microneedle array adopted deep reactive ion etching (DRIE) technology. The process flow was outlined as follows: a 4-inch single-side polished silicon wafer was putted into Piranha sulfuric acid liquor and boiled for 10–15 min till the Piranha liquor did not bubble. After cooled, Piranha liquor was poured and the chip was washed with deionized water for 5 min. The oxide layer on the silicon surface was about 2 µm thick by the oxidation process with alternating wet and dry oxygen. The polished side of the chip was coated with 6112D photoresist with the thickness of about 1.3 µm. The etched part was exposed through UV exposing and developing. The pentagram microneedle was formed by DRIE with the etching depth of about 250 µm. Poly(lactic-co-glycolic acid) (PLGA) nanoparticle (the average particle size 160.1±1.97 nm, PDI=0.073, self-made) wrapping Coumarin-6 was used as fluorescent mark to observe whether nanoparticle could penetrate stratum corneum by microneedle. The microneedle was stressed on the the stratum corneum of the human abdominal skin with a force of 15 N×1 min. Then the skin was situated in the transdermal diffusion pool with the stratum corneum side facing the supply pool. The phosphate buffer with fluorescent nanoparticles was poured into the supply pool for 24 h. Then the skin was taken out from the supply pool and cleaned the residue on the surface. To observe the distribution of nanoparticles in the skin, the skin was put on the glass slide under laser scanning confocal microscope (Leica TCS/SP, Germany) immediately. Taking the axle perpendicular to the skin stratum corneum as Z axle, scan the skin layer by layer along Z axle starting from the stratum corneum. Taking 488 nm as the excitation wavelength of Coumarin-6, observe the autofluorescence of the blank skin. Meanwhile the skin processed by microneedle was HE stained and observed under optical microscope.
ImmunityThe preparation of complexes was as follows: 250 µL of DNA vaccine solution (containing DNA vaccine 100 µg) was added to equivoluminal P123-PEI solution based on N/P=9 and mixed with vortexing. The mixed solution were incubated for 20 min at room temperature and then concentrated to approximately 50 µL by the ultra filtration membrane tube with molecular weight cutoff of 30 kDa.
The mice treated with microneedle were hair removed on the abdominal part with depilatory cream 48 h before the experiment, and the hairless area was 1.5×1.5 cm. In the experiment, the mice were fixed after anesthesia with pentobarbital sodium. The hairless area was processed with microneedle (to inject the microneedle into the skin and remove it after 2 min). DNA vaccine aqueous solution 50 µL (with 100 µg DNA vaccine) or P123-PEI/DNA vaccine complex 50 µL (with 100 µg DNA vaccine) per animal were uniformly painted to the microneedle-processed skin respectively used microsyringe. The mice were free from fixation 6 h later. Mice were also immunized by intramuscular injection of 100 µL PBS with 100 µg DNA or P123-PEI/DNA vaccine complex 50 µL (with 100 µg DNA vaccine) per mouse into quadriceps muscle, and mice with intact skin immunized by transcutaneous immunization of 100 µg DNA per mouse in 50 µL aqueous solution, and non-immune mice for comparison. The mice had been immunized twice at interval of 2 weeks.
Determination of Immunity ResponseBlood samples were collected from orbital posterior veins of the immunized mice at weeks 4, 6, 8, and 10 after primary immunization. The blood samples were allowed to clot and then centrifuged to separate serum. All the collected samples were preserved at −80°C until testing. The anti-HbcAg antibody was analyzed by ELISA kit. The splenocytes were separated from the immunized mice at week 7 after final immunization and cultured in vitro. The splenocytes were stimulated with rHBsAg protein (10 µg/mL). And the supernatants were harvested after 72 h and assayed the production of IFN-γ by ELISA kit.
Data StatisticsThe data was shown as mean±S.D. The statistical significance was determined by the analysis of variance (ANOVA) among ≧3 groups or Student t-test between 2 groups. In case of p values <0.05, it demonstrated the result was of significant difference.
The synthesis of the cationic polymer firstly protected one of P123 terminal hydroxyl groups with 4-methoxytriphenylmethyl chloride, and then the remaining free hydroxyl group was activated by succinimidyl carbonate. The activated hydroxyl group could be connected with the amino of PEI under mild conditions, and the monomethoxytrutyl protection group was removed with concentrated hydrochloric acid. The P123-PEI conjugate was separated from the non-conjugated P123 and PEI by gel permeation chromatography and then the cationic polymer P123-PEI were got. The specific course was shown in Fig. 1. The ratio of P123 and PEI of the cationic polymer P123-PEI was determined by calculating the integral values of the proton of –CH2CH2O– (P123) and that of –CH2CH2NH– (PEI) in 1H-NMR spectrum. Figure 2 showed the 1H-NMR spectrum of P123-PEI. The peaks position of the –CH2CH2O– proton was appeared at δ 3.6–3.9, and –CH2CH2NH– δ 2.65–3.40. Calculation showed that ratio of P123 to PEI was 1.05 : 1 in P123-PEI and molecular weight was 31.07 kd. The polydispersity coefficient was 1.37 which determined by Gel chromatography.
The N/P ratio, the ratio of concentration of total nitrogen atoms (N) of cationic polymer to the phosphate groups (P) of plasmid DNA, was the characteristic of the complex composition. P123-PEI/DNA complexes and PEI/DNA complexes of different N/P ratios were prepared and analyzed by agarose gel electrophoresis. The typical electrophoresis experiments results of all cationic polymers were showed in Fig. 3. It indicated that the move of plasmid DNA was gradually slowed down till completely blocked with the increase of cationic polymer (PEI 25 kd or P123-PEI), which illustrated that cationic polymer could neutralize the negative charge on DNA surface. When the N/P ratios exceeded the ratio of complete neutralization composition, plasmid DNA was entirely blocked and unable to move in gel electrophoresis, so cationic polymer and plasmid DNA formed stable complex. When N/P ratio was 2.5, PEI 25 kd and P123-PEI could totally block the move of plasmid DNA in gel electrophoresis, which meant that P123-PEI did not change the wrapping ability of PEI on DNA.
The particle size and the zeta potential of PEI/DNA complexes and P123-PEI/DNA complexes at different N/P ratios (6, 9, 12, 24, 48) were shown in Table 1. The average particle size of PEI/DNA complexes was less than 200 nm at N/P=6, while the particle size increased significantly at high N/P ratios. Especially at N/P=48, obvious aggregation and precipitation could be observed. The particle size of P123-PEI/DNA complexes decreased with the increase of N/P ratio and the average particle sizes were all less than 300 nm. Smaller particle size was good for complex to endocytose and transfect. Within the range of concentration studied, no aggregation and precipitation was observed at any N/P ratio for P123-PEI/DNA complexes. The existence of P123 was helpful for the colloid stability of the P123-PEI/DNA complexes. Zeta potential of complexes was closely related to their cellular uptake. The positive charge on complex surface facilitated cellular adsorption, but surely the complexes with strong positive charge would cause a great quantity of cellular death. According to Table 1, with the increase of N/P ratio, the zeta potential of complexes also increased, which indicated that the positive charge of amino group of cationic polymer gradually neutralized the negative charge of DNA phosphoric acid group. The zeta potential measurements were consistent with the results of gel electrophoresis experiments. At the same N/P ratio, the zeta potentials of P123-PEI/DNA complexes were all significantly lower than that of PEI/DNA complexes. Thus, P123 can noticeably change the surface charge distribution of the complexes after modifying.
| N/P | Particle size | Zeta potential | ||
|---|---|---|---|---|
| PEI 25 kd | P123-PEI | PEI 25 kd | P123-PEI | |
| 6 | 164±31 | 268±15 | 22.4±1.7 | 18.4±1.3 |
| 9 | 121±22 | 194±10 | 30.4±2.7 | 22.8±2.2 |
| 12 | 106±6 | 164±11 | 39.7±2.5 | 25.7±0.8 |
| 24 | 268±41 | 125±8 | 42.3±0.7 | 30.5±1.6 |
| 48 | 8521±839 | 101±6 | 47.9±1.1 | 36.7±0.5 |
The results of the cellular transfection and toxicity of PEI/DNA complexes and P123-PEI/DNA complexes at different N/P ratio (3, 6, 9, 12, 18) could be referred to Fig. 4. Within the N/P ratios range determined, the transfection efficiency of PEI/DNA complex sharply declined with the increase of N/P ratios (>3), and the best transfection efficiency appeared at N/P=6. The transfection efficiency of P123-PEI/DNA complex was best at N/P=9. With the increase of N/P ratio (>6), the transfection efficiency of P123-PEI/DNA complex was showed little change and all significantly higher than the best transfection efficiency of PEI/DNA complex (p<0.001). It could be seen from Fig. 4b that the cellular viability of P123-PEI/DNA complex was all significantly higher than that of PEI/DNA complex within N/P ratio range determined. When N/P ratio was larger than 9, the toxicity of PEI/DNA complex suddenly increased, and only about 50% cells survived at N/P=18. However, above 70% cells survived when N/P ratio of P123-PEI/DNA complex was 18. So it was possible that the comparatively higher cytotoxicity of PEI/DNA complex at higher N/P ratio (12, 18) was the reason for its lower transfection efficiency.
Values represent mean±S.D. (n=3). (a) Significantly different between P123-PEI (N/P=9, or 12, or 18) and PEI 25 kd (N/P=6); ◆ p<0.001. (b) Significantly different between P123-PEI and PEI 25 kd at same N/P value; * p<0.05, ▲ p<0.01, ◆ p<0.001.
DRIE was adopted to prepare pentagram microneedle in our study. The stereoscan photograph of microneedle arrays was showed in Fig. 5. 10×10 microneedle arrays were prepared on the silicon chip with an area of 0.25 cm2. The length of single microneedle was 200 µm.
Isolated skin processed by microneedle was made frozen section and observed by the optical microscope after HE staining. As shown in Fig. 6, the thickness of epidermis of the skin was approximately 50 µm. The epidermis of the skin could be penetrated by the microneedle and formed mircopores which could provide channels for nanoparticle with diameter of approximately 20–30 µm to enter the skin. Confocal microscope was used to observe the fluorescence nanoparticle to penetrate the epidermis of the skin. As shown in Fig. 7, blank skin had almost no spontaneous fluorescence under 488 nm exciting light and did not disturb the observation for fluorescence nanoparticle. The skin could be permeated by fluorescence nanoparticle through the micropores caused by penetration of microneedle in the skin. The deeper the depth of the skin was, the less the amount of nanoparticle was observed. Nanoparticle could still be observed at 68.32 µm, indicated that nanoparticle had already permeated into the layer of dermis. In the control group on blank skin (without being processed by microneedle), fluorescence could be observed as well. However, the fluorescence signal was much less than the skin processed by microneedle within the same sight range. It showed that although nanoparicle can not enter the skin by penetrating the barrier of stratum corneum, through the hair follicle existing in the skin, small amount of nanoparticle could enter the skin.
Skin was HE stained after 1 h processed by microneedle.
(A) Image of the skin in the microneedle group by the fluorescence microscope (×4 objective). (B) Image of the blank human abdominal skin scanned at 488 nm excitation wavelength by CLSM. Images of skin at different depth in the microneedle group scanned at 488 nm excitation wavelength by CLSM (×10 objective): (C) 0 µm (D) 34.16 µm (E) 68.32 µm. Images of skin at different depths in the control group scanned at 488 nm excitation wavelength by CLSM (×10 objective): (F) 0 µm (G) 68.32 µm (H) 136.64 µm.
Under the same immunizing dose, at the 4th, 6th, 8th, and 10th week after first immunity, ELISA was used to determine the anti-HbsAg of serum of the immunized mouse. The result was shown in Fig. 8. At all time points, immunoglobulin G (IgG) antibody titer in the serum of mice immunized percutaneously by microneedle and intramuscularly were significantly higher than that of control group (non-immune and DNA intact skin, p<0.001). Mice immunized percutaneously with P123-PEI/DNA complexes by microneedle, compared with mice immunized percutaneously with naked DNA by microneedle and intramuscular directly or the complexes intramuscular directly, was shown remarkable increase in IgG antibody titer in the serum (p<0.001). The effect of immunity with naked DNA vaccine by microneedle was higher than that of intramuscular (p<0.05), and the effect of immunity with naked DNA vaccine by microneedle was same to that of the complexes intramuscular directly (p>0.05). The result demonstrated that the complex can markedly improve the humoral immunity level of DNA vaccine after immunization percutaneously by microneedle. Splenocyte of mice was cultured in vitro and the amounts of IFN-γ secretion from splenocyte was determined by ELISA. The amount of IFN-γ of P123-PEI/DNA complexes microneedle group was 4.7 times higher than that of DNA vaccine intramuscular group and 2.6 times higher than that of naked DNA vaccine microneedle group and 3.18 times higher than that of the complexes intramuscular directly, significantly higher than that of DNA vaccine intramuscular group and naked DNA vaccine microneedle group and the complexes intramuscular group (p<0.001), and the result was shown in Fig. 9. The amount of IFN-γ of naked DNA vaccine microneedle group is significantly higher than that of DNA vaccine intramuscular group (p<0.01). The amount of IFN-γ of the complexes intramuscular group has nonsignificant difference to DNA vaccine intramuscular group and naked DNA vaccine microneedle group (p>0.05). The result demonstrates that percutaneous immunity with microneedle is an advantageous manner to improve effect of DNA vaccine cellular immunity.
Values represent mean±S.D. (n=8) of log 10 of the reciprocal end-point serial different fold serum dilutions required for OD readings to reach a value of about 0.500. Mice immunized percutaneously with P123-PEI/DNA complexes by microneedle (Complex microneedle) or naked DNA by microneedle (DNA microneedle), and immunized intramuscularly with P123-PEI/DNA complexes (Complex IM) or naked DNA (DNA IM); mice with intact skin immunized percutaneously by naked DNA (DNA intact skin) and non-immune mice for comparison. Compared with DNA intramuscular group, * p<0.05, ▲ p<0.01, ◆ p<0.001.
Splenocytes were obtained from six mice per group at week 7 after the final immunizations with different formulations. Pooled splenocytes from each group were stimulated with rHBsAg (10 µg/mL) overnight. The supernatants were harvested after 72 h and assayed for the production of IFN-γ by ELISA. Values represent mean±S.D. (n=8). Mice immunized percutaneously with P123-PEI/DNA complexes by microneedle (Complex microneedle) or naked DNA by microneedle (DNA microneedle), and immunized intramuscularly with P123-PEI/DNA complexes (Complex IM) or naked DNA (DNA IM); Mice with intact skin immunized percutaneously by naked DNA (DNA intact skin) and non-immune mice for comparison. Compared with DNA intramuscular group,▲ p<0.01, ◆ p<0.001.
DNA vaccines, which could induce a wide range of humoral immunity and cellular immunity, show promising development prospect. As of today, only three kinds of DNA vaccines—Horse West Nile virus, salmon infectious and hemorrhagic necrosis virus and canine melanoma vaccine—were allowed in the market.19) The DNA vaccines which target to deal with the illnesses such as malaria, hepatitis B and human immunodeficiency virus have entered clinical experimental stage.19) However the naked DNA vaccine would be easily degraded by nuclease. Moreover, the histocyte’s uptaking is minimal due to its physicochemical property characters; especially uptake efficiency of APCs was extremely low, which is the reason for the low immune level of the DNA vaccines.2) To improve the transfection efficiency of the DNA vaccine, a non-viral carrier of the DNA vaccine was prepared in this experiment and PEI was modified to be the carrier of the DNA vaccine. In order to improve the defects of PEI which had high cytotoxicity and poor colloid stability, Pluronic P123 was used to modify PEI. Some articles reported that hydrophilic polymers such as PEG were used to modify PEI to improve the stability of PEI/DNA complex and reduce the cytotoxicity. The less cytotoxic PEI derivatives, however, showed markedly reduced gene transfection efficiency compared to those unmodified PEI due to decreased cellular association and internalization.20) PEI modified by P123 can not only reduce the cytotoxicity of the PEI remarkably, but also keep high transfection efficiency of the complex, because P123 contained hydrophobic PPO chains and hydrophilic PEO chains. The “stealth” effect of the PEO chains can reduce the cytotoxicity of P123-PEI/DNA complex and improve their colloidal stability, while the PPO chains can enhance the lipophilicity of the complex and maintain the affinity between the complexes and cell membranes, which was conducive to cell endocytosis and can maintain high transfection efficiency. Sriadibhatla et al. reported that Pluronic can activate the relevant selective signaling pathways and upregulate the transcription of genes, change the biological responses of the gene expression, resulting in an enhancement of gene expression.21) Our research group in the early experiment used different amount of different type of Pluronic to modify high molecular weight PEI, and obtained a series of Pluronic-PEI.22) The degree of Pluronic and the Pluronic type of Pluronic-PEI had a significant effect on the Pluronic-PEI transfection efficiency. The polymer synthesized by Pluronic and PEI at the molar rate of 1 : 1 retained higher transfection efficiency. The PEO chains of the Pluronic molecules should not be too long and needed to have some certain lipophilicity to maintain the high transfection efficiency of the Pluronic-PEI. Pluronic P123 with 1 : 1 molar weight in this study was chosen to modify the high molecular weight PEI. The experimental results showed that P123 was able to improve the colloidal stability of the complex and avoid aggregation and precipitation of the complex at high N/P ratio. The PEI modifying by P123 can also reduce the cytotoxicity of PEI significantly. The optimal transfection efficiency (N/P=9) of P123-PEI was also significantly higher than that of unmodified PEI.
Due to the barrier effect of the stratum corneum, DNA vaccine was unable to go to the active epidermal layer through stratum corneum according to the conventional chemical transdermal method. Equally the intact skin can not be penetrated by nanoparticles, such as cationic polymer/DNA complex, excluding through hair follicle. A safe, simple, and painless delivery method was provided by microneedle with which the macromolecular drugs, especially the nanoparticles, can easily penetrate through the stratum corneum barrier. This pentagram microneedle array were prepared in this study and the puncture experiments of isolated skin showed that a hole with diameter about 20–30 µm can be formed on the surface layer of skin processed by microneedle (Fig. 6).
The study used the PLGA nanoparticles wrapped the fluorescent probe (Coumarin-6) which simulate the DNA vaccine/P123-PEI complex to penetrate through these holes to the active epidermal layer. The average diameter of the prepared PLGA nanoparticles was 160.1 nm which was similar to the diameter of the DNA vaccine/P123-PEI complex (N/P=9). The Coumarin-6 wrapped by the PLGA nanoparticles was not released in the transdermal experiment (data not shown). The distribution of the nanoparticles in the skin can be observed relying on the confocal microscope, due to the high fluorescence efficiency of the Coumarin-6. The experimental results showed that there were no interference on the autofluorescence of the skin and the nanoparticles can enter the skin through the holes caused by the microneedle. With the increasing of the skin depth, the fluorescence intensity decreased. The nanoparticles were mainly in the skin epidermis, and rare were in dermis. No nanoparticle can reach to the receiving liquid through the skin (data not shown). As the length of microneedles was 200 µm, only the epidermis and a few of the dermis could be punctured. But the whole skin would not be punctured. The required piercing strength and the penetrating depth of the microneedles were closely related to the size and interval of the microneedles.23) The holes formed by the experimental microneedles were more than 20 microns, which could provide enough room for nanoparticle to pass through. Some articles reported that nanoparticle can enter into the intact skin through hair follicle,24,25) and it was also found that a small quantity of nanoparticle entered into the intact skin through the cutaneous appendages in this experiment. But the amount was much less than that through the skin processed by the microneedles. The PLGA nanoparticles used in this study were negatively charge with a zeta potential value of −30 mV, while most of the zeta potential of the DNA vaccine/P123–PEI complexes were above +20 mV. Although the diffusion of emulsions through the skin can be significantly influenced by the surface charge,26) the diameter of the nanoparticle was observed as the main influential factor for its penetration with microneedle.27) So the PLGA nanoparticles having the similar diameter scope with the complexes were chosen as a size-representative model for the DNA vaccine/P123–PEI complex to diffuse through the skin. Charbri et al.28) reported that the polystyrene nanospheres with the diameter of about 110 nm was used to simulate plasmid DNA/cationic liposome complexes with the same diameter studying the diffusion through the skin treated by the microneedles. The results showed that polystyrene nanospheres can enter to the accept phrase through the skin treated by the microneedles and reach the saturation at 12 h.
It was widely accepted that naked DNA was usually injected by the intramuscular route. DNA was taken up by myocytes, leading to immune response as cytotoxic T-lymphocyte. However, uptaking of DNA by myocytes was minimal and only a minor fraction of the cells were involved. Moreover, because MHC class I molecules were not professional antigen-presenting cells lack of the abililty to excrete vital co-stimulatory molecules, although it can be carried by myocytes, the immune effect was low.29) On the contrary, the skin is proved to be an attractive site for effective expression of antigen, because there is a large quantity of APCs in the viable epidermis of the skin which could product strong humoral immunity and cellular immunity. The DNA vaccine can not be delivered to the viable epidermis effectively, so the viable epidermis could not be a suitable vaccine inoculation site. Adhesive tapes were used to strip the stratum corneum again and again in some researches, then the viable epidermis layer was exposed in order to inoculate DNA vaccine.30) Compared to adhesive tapes, microneedle penetration is a minimal invasive approach, which makes only a little hurt to the skin. The micropores to be penetrated by the microneedles is temporary and has normal physiological function of the skin. In this study, microneedles were used to locate the viable epidermis layer of skin with the complex formed by cationic polymer P123-PEI. Meanwhile DNA vaccine with the diameter of about 200 nm was used to enhance the transfection efficiency of the DNA vaccine. It showed that the microchannel formed by the microneedle could help the complex enter into the viable epidermis easily.
The experimental results showed that antibody titer in the serum of mice immunized percutaneously with the complex by microneedle was improved significantly than that of mice immunized percutaneously with naked DNA vaccine by microneedle and immunized intramuscularly with naked DNA vaccine or immunized intramuscularly with the complex. The rationale for that maybe because the DNA vaccine complex captured by the APCs in the viable epidermis can transfect and expresse antigen efficiently, thereby produce stronger humoral immunity effect. The experimental results showed that the amounts of IFN-γ secreted from isolated splenocyte of the complex microneedle group after stimulating by the antigenic was higher than other groups, which illustrated that it could produce stronger cell immunity effects.
Silicon microneedle was prepared in the study to penetrate the skin into the viable epidermis. The micropores were formed with certain size on the surface layer of skin to help the nanoparticles with particle size of approximately 200 nm reach the viable epidermis.
Genetic vector P123-PEI of high efficiency and low cytotoxicity was synthesized with DNA vaccine to form nano-complex particle size of which was approximately 200 nm, so that it could help the cells of the viable epidermis uptake DNA vaccine efficiently. The mice were immunized percutaneously with DNA vaccine nano-complex through the abdominal skin processed by microneedle. The result showed that the level of humoral and cellar immunity of the mice was improved significantly. All that proved that immunization percutaneously with DNA vaccine/cationic polymer nano-complex by microneedle array was a kind of effective and promising DNA vaccine immune method.
This study was funded by No. 51 China Postdoctoral Sustentation Fund, Medical Fund of Zhejiang Province (No. 2012KYB213) and Medical Fund of Lanzhou Military Region of PLA (No. CLZ12JA11).
