TWI286076B - Block copolymers and nano micelles comprising the same - Google Patents

Abstract

A block copolymer. The block copolymer includes A block and B block, wherein the A block represents a polyamide and the B block represents a copolymer including a polyethylenimine and a polyamide. The invention also provides a nano micelle including the block copolymer.

Description

[Technical Field] The present invention relates to a polymer, and particularly relates to a group copolymerized south molecule and a nanocapsule comprising the copolymerized south molecule. [Prior Art] In recent years, acid-base responsive polymer micelles have been a hot research field, but most of them focus on physical properties. The design principle is usually to add a polyelectrolyte to the polymer, that is, an ionic polymer, which changes the configuration of the microcells by the change of affinity, hydrophobicity or charge before and after dissociation of the φ polyelectrolyte. Diethylaminoethyl-glucan (diethyhminoethy Kdextran, DEAE-dextran) was the first cationic south molecule for gene therapy. Yamaoka et al. found charged functional diethylaminoethyl (DEAE) and dextran (dextran). The ratio of polycomplex affects gene transfection. The results show that the cytotoxicity of diethylaminoethyl-glucan decreases with the decrease of diethylaminoethyl content. The transfection effect also deteriorated (Chem. Lett. 11, 1171, (1998)). However, diethylaminoethyl-glucan has low transfection efficiency, high cytotoxicity, and is not biodegradable, which limits its practical application. Yamaoka et al. have compared the gene expression of many different cationic polymers, and found that the aggregates formed by these polymers and DNA are very similar in volume or interface potential, but only have hydroxyl groups. The polymer genes of 〇xyl group), amide group, tertiary amine and quaternary amine are particularly effective. It is generally believed that the hydrophilicity of the hydroxyl group and the guanamine group allows the polycomplex to avoid 0424-A21354TWF(N2); P02940056TW; david 5 1286076 Λ f * «free aggregation and facilitate DNA dissociation.

Alonso et al. prepared a poly(lactic acid)-p〇ly (ethylene glycol) chain copolymer and coated DNA as a gene carrier. This polymer has no positive charge, so it is coated with water/oil-water emulsification method (J· Control·

Release, 75, 211. (2001)) 〇

Lemieux coated DNA with Pluronic® copolymer polymer, which has no significant effect in vitro. With intramuscular injection, the transfection efficiency is about 5 to 20 times higher than that of direct DNA (Gene Ther·, 7, 986 (2000). )).

Jeong et al. used poly(d5l-lactic-co-glycolic acid, PLGA) to bond with oligonucleotide (ODN) to form polylactic acid-glycolic acid in an aqueous environment. The microcell structure of the oligonucleic acid (Bioconjug. Chem., 12, 917 (2001)). It takes a long time to release the oligonucleic acid by degradation of polylactic acid-glycolic acid, which is a sustained release. Oligonucleic acids are often antisense DNA fragments that, when introduced into a cell, bind to DNA and block their function and inhibit protein expression.

Maruyama coats DNA with poly(lactic acid), PLA, and uses the electrostatic force of poly(L-lysine)-graft-polysaccharide to modify the outer layer. After the outer layer is coated with polysaccharide, the protein is adsorbed. The phenomenon of aggregation can be effectively improved (Bioconjug. Chem., 8, 735 (1997)"

Kataoka et al. synthesized poly(ethylene glycol-&-polyethylenimine, PEG-PEI) copolymer chain using line 0424-A21354TWF(N2); P02940056TW; david 6 1286076 1 ' • 6-polyethyleneimine coated DNA, hydrophilic polyethylene glycol protects the polyplex in the outer layer (Macromolecules, 33, 5841 (2000)). However, the experimental results show that polyethylene glycol can reduce the transfection effect of polyethyleneimine without receptor-mediated endocytosis (Gene Ther·, 7, 126 (2000)).

Petersen et al. used a series of polyethylene glycol-polyethyleneimine (poly(ethylene glycol)-g-polyethylenimine, • PEG_g- with different molecular weight and grafting degree of poly(ethylene glycol). PEI) was the subject of investigation (Bioconj. Chem., 13, 845, (2002)). As a result, it was found that, besides the copolymer having a polyethylene glycol molecular weight of less than 5 kDa, the particle size of the polycomposite was quite stable at an ionic strength of 0.15 M sodium chloride, and the red blood cell agglutination was reduced. With the increase of the molecular weight and grafting degree of polyethylene glycol, the interface potential will gradually decrease. In addition, polyethylene glycols of different molecular weights have little effect on the DNA coating ability of polyethyleneimine, but polyethylene glycol-g-polyethylene is compared with polyethyleneimine which is not grafted with polyethylene glycol. The amine requires a greater amount to coat the DNA, indicating that the polyethylene glycol hinders the binding of the polyethyleneimine to the DNA. In the test of cells, when the grafting rate of polyethylene glycol is increased, the toxicity to cells is lower, but the transfection efficiency is also lowered. Frisch et al. used polyethylene glycol/linear polyethyleneimine (p〇ly(ethylene glycol)/L_polyethylenimine, PEG/L-PEI) copolymer chain as the gene carrier, which also obtained water solubility, cytotoxicity and transfection. The result of reduced efficiency (J Control. Release, 81, 379, (2002)). Therefore, the hydrophilic polymer polyethylene glycol is used to change the cationic polymer to 0424-A21354TWF(N2); P02940056TW; david 7 1286076.*. , which can increase the hydrophilicity of the polycomplex and mask the positive charge of the cationic polymer. The effect of stabilizing the polycomplex is achieved, and in addition, the cytotoxicity of the cationic polymer can be reduced. However, for polyethyleneimine, the poor transfection efficiency of polyethylene glycol manganese polyethyleneimine/L_p〇lyethylenimine, PEG/L-PEI) copolymers still limits its application value. SUMMARY OF THE INVENTION The present invention provides a group copolymerized polymer comprising a segment A and a segment B wherein the A segment is a polyamine amine B segment is a copolymer of polyethyleneimine and polybrene. The present invention further provides a nano-cell, comprising a plurality of agglomerated copolymers and a DNA fragment, which are described in the inside of the micelle. [Embodiment] The present invention provides a cluster copolymerized polymer comprising an A segment and an 8-strand segment. The A segment in the structure may be a polyamine, the B segment may be a copolymer of polyethyleneimine and polyamine, and the B segment may be positively charged in a neutral aqueous environment. • The group-copolymerized polymer may be a diblock copolymerized polymer, and the polyamine in the structure may include polyoxazoline (p〇ly (〇xaz〇line), ?〇2) or a derivative thereof The polyethyleneimine may be a linear or branched polyethyleneimine, and a linear polyethyleneimine is preferred. The copolymerized polymer of the present invention can be represented by the following chemical formula (1). L+g - CH2-CH2j7D+CH2CH2.NHiCH2.CH2—j^L , , Π (I) In the formula (I), L may be a starter for ring-opening polymerization, such as nitrogen or an alkyl group, preferably Is an alkyl group, Z may be hydrogen or C2 2i fluorenyl group, preferably fluorenyl group, 0424-A21354TWF(N2); P02940056TW; david 8 1286076 1庐·D may be sulfur-sulfur, sulfhydryl or self-priming Preferably, the sulfur-sulfur bond, χ, m and η may be an integer from 1 to 10,000, and x/(m+n) may be 〇~5. The present invention further provides a nanocapsule comprising a plurality of the above-mentioned copolymerized polymer and a DNA fragment coated inside the microcapsule. The chain copolymerization The weight ratio of the polymer to the DNA fragment is generally between 1: 1 〇〇 and 1 〇〇 : 1. In the microcell, the positively charged B segment of the cluster copolymer is bonded to the negatively charged DNA fragment by the electrostatic force between the positive and negative charges, and the A segment is exposed to the outside of the cell. In this way, the inside of the Xinnai cell is hydrophobic, the exterior is hydrophilic, the particle size is generally between 10 and 1,000 nm, and the interface potential is generally between -50 and 50 mV. Since the nano-cells of the present invention are electrically neutral, sputum can smoothly enter the cells through the cell membrane, and when the micelles are in an acidic environment, for example, a pH lower than 5.5 ′ will disintegrate spontaneously and release DNA for transcription. Translation work. The present invention contemplates an acid-base response type such as polyoxazoline-polyoxazoline/polyethyleneimine (p〇ly(〇xazoline)_6_p〇ly(oxaz〇iine/ethylenimine), • P〇z-6-P The (Oz/EI)) group chain copolymer is used as a smart gene carrier, which utilizes changes in the liquid phase and intracellular pH to control and help gene release. Please refer to Figure 1 for the process by which the copolymer of the cluster is combined with the gene to form the micelle. The group copolymer 1 is divided into, for example, a chain segment of a hydrophilic polyoxazoline and a B segment of a polyoxazoline/polyethyleneimine copolymer, in a neutral aqueous phase, a copolymer of a chain copolymer The B segment will bind to the gene 2 to form an a-segment, the B-segment and the gene 2, and the c〇re-shell structure 3 〇0424-A21354TWF(N2); P02940056TW; david 9 1286076 , * · * After the environmental acid test value decreases, the polyoxazole U forest of the above A segment will generate intramolecular and intermolecular hydrogen bonds to aggregate and shrink, causing damage to the microcell structure 3 Release gene 2. This special mechanism controls the release of genes into cells for high transfection and therapeutic effects. At the same time, by adjusting the degree of proper hydrolysis, the polyoxazoline/polyethyleneimine of the above B segment can also provide a hydrophilic force to assist gene desorption, all of which contribute to gene delivery and release. Further, the polymer used in the present invention, in addition to having acid-base sensitivity, is involved in conventional gene carrier materials such as polylactic acid-polyethylene glycol (PLA-PEG), polylactic acid glycolic acid-oligonucleic acid (PLGA). -ODN), polyethylene glycol-polyethyleneimine (PEG-PEI) or polyethylene glycol-g-polyethyleneimine (PEG-g-PEI), also has low toxicity and good biocompatibility . The copolymerized chain polymer of the present invention can be synthesized in the following manner. First, using a monofunctional starter such as methyl j^-toluenesulfonate (MeOTs), methyl methanesulfonate (MeOMs) or iodoethane (Mel) for Φ, for example, 2· The indoleamine monomer of 2-ethyl-2-oxazoline is subjected to cationic ring-opening polymerization. After the reaction is terminated, it is cooled to room temperature and then a solid such as potassium thioacetate (KSAc) is added thereto, and the terminal is introduced into, for example, thioacetate (SAc). Thereafter, the obtained polyoxazoline (POz-SAc) having a terminal thioacetic acid group is reacted with, for example, a 2,2 dithiol (2,2'-dithiodipyridine) P-pyridine compound to obtain, for example, polyfluorene. An intermediate product of oxazoline-pyridine (POz-Py), after which the intermediate product containing the butyl structure is placed on 0424-A21354TWF(N2); P02940056TW; david 10 1286076 -, 1 ^ For example, an acidic solution of hydrochloric acid is subjected to hydrolysis to obtain, for example, a hydrolysis product of poly(oxazoline-co-ethylenimine)-pyridine, P(Oz/EI)-Py. The hydrolyzed product is then reacted with, for example, a polyoxazoline (POz-SAc) having a terminal thioacetic acid group to obtain, for example, a polyoxazoline-polyoxazoline/polyethyleneimine (P0z-bP (0z/). EI)) agglomerate copolymer. The features and advantages of the present invention will be further illustrated by the following examples.

[Examples] [Example 1] Poly-2-ethyl-2-oxazoline-poly(2-ethyl-2-oxazoline/ethyleneimine) (PEOz-b-P(EOz/EI)) synthesis

(1) Synthesis of poly-2-ethyl-2-oxazoline-thioacetic acid (corporate quinone-2-like c)

Kscoch3 o°c ο II S-c-CHq

Er First, mix 370 mg of methyl-toluenesulfonate (MeOTs) (1.98 mmol) with 20 ml of 2 ethyl-2-oxazoline (2_ethyl-2_oxazoline, ΕΟζ) (197·72 mmol) On 60 0424-A21354TWF (N2); P02940056TW; david 11 1286076 «Qin» 1 ml of anhydrous acetonitrile solvent to prepare a mixed solution. Next, the mixed solution was heated to 100 ° C under a nitrogen atmosphere and stirred for 24 hours to carry out a polymer polymerization reaction. Thereafter, 0.7 g of potassium thioacetate was added at 0 ° C to terminate the above PEOz polymer polymerization. After stirring the solution for 24 hours, it was filtered with hydrazine and purified by diethyl ether precipitation. After complete drying, a brown powder of PEOz-SAc product was obtained. The molecular weight and polydispersity of PEOz-SAc polymers can be determined by gel permeation (gel permeation).

Chromatography, GPC) analysis obtained. (2) Synthesis of poly-2-ethyl-2-oxazoline-B-pyridinium H3Sn^; >-C-ch3

NH3/MeOH R.T. (3) Synthesis of poly(2·ethyloxazoline ethyleneimine)-indolepyridinium (iY 〇7)

First, 1 gram of PEOz-SAc (0.1 mmol) was mixed with 1 mM of 2,2,-dithiodipyridine (〇.5 mmol) in 20 liters of ammonia/sterol. Solution (7N). Next, the mixed solution was stirred for 24 hours under a nitrogen atmosphere at room temperature to remove the solvent. The residue remaining was redissolved in dichloromethane and precipitated several times with diethyl ether. Thereafter, the obtained product was purified by vacuum drying to obtain a yellow powder of PE0z-Py. Next, 1 g of PEOz-Py was added to a mixed solution of 1 ml of concentrated hydrochloric acid (35%) and 8 ml of water and heated to Celsius 1. The temperature lasts for 3 hours. 0424-A21354TWF(N2); P02940056TW; david 12 1286076 .. * » After that, sodium hydroxide was added to adjust the pH of the solution to 9-10. The product was purified by dialysis for 3 days. After being completely dried, a viscous semi-solid P(EOz-EI)-Py product was obtained. (4) Synthesis of poly-2-ethyl-2-oxazoline-poly(2-ethyl-2-oxazoline/ethyleneimine) (PEOz-b-P(EOz/EI))

First, 1 gram of P(EOz-EI)-Py (0.196 mmol) and 235 mg of PEOz-SAc (0.065 mmol) were mixed in 15 ml of ammonia/methanol mixed solution (7N) under nitrogen atmosphere at room temperature. The reaction was carried out for 24 hours. The product was dialyzed against decyl alcohol and distilled water for 3 days to remove unreacted P(EOz-EI)-Py and other by-products. After the evapotranspiration step, the final product PEOz-b-P (EOz/EI) is obtained. The polymer PEOz_b-P (EOz/EI) was stored dry at -20 ° C before use. [Example 2] Preparation of micelles First, 5 pg of DNA was taken with poly-2-ethyl-2-oxazoline-poly(2-ethyl-241 oxazoline/ethyleneimine) (PEOz-bP (EOz/) EI)) The copolymerized polymer is mixed, and the weight ratio of the polymer to the DNA is 込1 to 20 0. After uniformly mixing, a DNA-coated micelle is obtained by reacting at room temperature for 30 minutes. The gene selected in the present embodiment is a plastid DNA of a luciferase-containing gene, and a PUHC 13-3", which belongs to a reporter gene. 0424-A21354TWF(N2); P02940056TW ; david 13 1286076 .* 1 > [Example 3] Cellular particle size analysis The cell solution was placed in an acrylic container to measure the cell size. For the results, please refer to Fig. 2 'Illustration of poly(2-ethyl-) 2-?. Lin/Ethyleneimine) (P(EOz/EI)) complex with poly-2-ethyl-2-oxazoline-poly(2_ethyl_2_oxazoline/ethylene Particle size change of the PEOz-bP(EOz/EI) complex in different solutions, wherein the poly(2-ethyl-2-oxazoline/ethyleneimine) complex includes B-PEI, L -76 and L-89, poly 2-ethyl samarium salicin (2-ethylglycanoxazoline / ethyleneimine) complexes including 4k-76 and 4k-89. The results can be seen, regardless of the poly (2-ethyl-2-oxazolene/ethylene subfec) (P(EOz/EI)) complex or poly-2-ethyl-2-indole _ poly(2_ethyl_2 port The oxazoline/ethyleneimine) (PEOz-b_P(EOz/EI)) complex has a particle size of approximately 200 nm in water. When the ion concentration in the solution is increased to 〇15M The poly(2-ethyl-2-oxazoline/ethyleneimine) complex will increase the particle size by 2 to 3 times due to agglutination, but poly-2-ethyl-2-oxazoline poly(2-B) The particle size of the base 2_carbazide/ethyleneimine complex is not much different from that in pure water, and the poly-2-ethyl-2-11⁄2唆(唆) which shows hydrophilicity is indeed helpful. The stability of the polycomplex is stable. [Example 4] For the analysis of the cell potential, please refer to Figure 3 for the poly(2-ethyl-2-oxazolidine/ethyleneimine) (Ρ(ΕΟζ/ΕΙ) The interface potential measured by the complex and poly-2-ethyl-2-antimonin-poly(2_ethyl port μ σ sitin/ethyleneimine) (PEOz-bP(EOz/EI)) complex The difference 'in the poly(2-ethyl-2_曙嗤琳/ethyleneimine) complex includes 0424-A21354TWF(N2); P02940056TW; david 14 1286076 .» 1 * B-PEI, L-76 and L -89, poly-2-ethyl-2-salin-poly(2-ethyl-2-oxooxazoline/ethyleneimine) complexes including 4k-76 and 4k-89. , poly(2-ethyl-2,oxazoline/ethyleneimine) (P(EOz/EI)) complex with a positive charge, and poly-2-ethyl-2-oxazole-poly (2-B Base_2_曙曙琳/ethyleneimine)(p The EOz-b-P(EOz/EI)) complex is close to electrical neutrality. [Example 5] Analysis of microcellular acid-base response • Please refer to Figures 4 to 6 for poly(2-ethyl-2-oxazoline/ethyleneimine) (P(EOz/EI)) complex and poly 2-ethyl·2_oxazoline-poly(2-ethylmoxazoline/ethyleneimine) (PEOz-bP(EOz/EI)) complex in the acidity response of various ρΗ値' 2-Ethyl-2-hydrazine/ethyleneimine) complex is LI-76 ' 1 2-ethyl 2 - 〇 唾 唾 - - poly (2-ethyl _2 - 曙 琳 / ethene The imine) complexes include 4k-76 and 4k_89. From the electropherogram, we can observe the poly(2-ethyl-2_卩萼卩萼琳-poly(2-ethyl_2«•oxaxazoline/ethyleneimine) (PEOz-bP(EOz/EI)) complex. It is very stable in a neutral environment. However, when the pH value drops to 5.5, 5 or 4.5, a clear strip pattern appears in the figure, which means that the poly complex has dissociated and released DNA (5th, ό Figure). On the same day, the poly(2-ethyl-2-indene/ethyleneimine) (p(e〇z/ei)) complex was tested and found to be poly(2-ethyl-2-anthracene). /Ethymidine imine) complex is quite stable under different J clothing brothers (Fig. 4). Therefore, it can be inferred that by the acid-base reaction of the poly-2-ethyl-2-oxazoline (PE〇z) polymer, it is possible to change the structure of the poly complex to release the contained nucleic acid. [Example 6] 0424-A21354TWF(N2); P02940056TW; david 15 1286076 .' ι . Microcytotoxicity test First, human diploid fibroblast was cultured at 37 ° C, carbon dioxide concentration 5%. In a constant temperature incubator, the growth was observed with an inverted microscope. After the cells are up to eight minutes long, use the 0.25% trypsin-EDTA cleaning solution to wash the cells in the dish, mix the cell stain trypan blue, and use the cells under the microscope. The counter counts the number of cells. 1 x 10 4 cells were seeded per well in a 96-well culture dish. After 24 hours, remove the medium • and add the poly(2-ethyl-2-oxazoline/ethyleneimine) (Ρ(ΕΟζ/ΕΙ)) complex with poly-2-ethyl-2-hydrazine a concentration of oxazoline-poly(2-ethyl-2-oxazoline/ethyleneimine) (PEOz-bP(EOz/EI)) complex at concentrations of 0.001, 0.01, 0.1, 1 and 10 mg, respectively /ml,. After a certain period of time, fresh medium was replaced and ΙΟμΕ in MTT (5 mg/mL in PBS) was added for 4 hours. The medium was again removed, and 100 μί of dimethyl sulfoxide was added to dissolve the crystals. After reacting for 12 hours at room temperature, the absorption at a wavelength of 570 nm was read with a 96-well plate enzyme reader. The experiment was repeated 6 times (η = 6), and the cell viability was calculated as: cell viability (%) = absorbance sample / absorbance positive control experimental results found that poly 2-ethyl-2 -Oxazoline-poly(2-ethyl-2-oxazoline/ethyleneimine) (PEOz-bP(EOz/EI)) complex is significantly more toxic than poly(2-ethyl-2-oxazoline) /Ethyleneimine) (P(EOz/EI)) complex is low, such as poly-2-ethyl-2-oxazoline-poly(2-ethyl-2-oxazoline/ethyleneimine) complex At a concentration of 1 mg/ml, the cell viability of 60% or more can still be maintained. Please refer to Fig. 7, where the poly(2-ethyl-2-oxazoline/ethyleneimine) complex includes 0424-A21354TWF (N2). ); P02940056TW; david 16 1286076 .* 1 > Β·ΡΕΙ, L-76 and L-89, poly-2-ethyl-2-oxazoline-poly(2-ethyl-2-porto-oxazoline/ The ethyleneimine) complexes include 4k-76 and 4k-89. While the present invention has been described above by way of a preferred embodiment, it is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

0424-A21354TWF(N2); P02940056TW; david 17 1286076 & ft 4 · [Simplified Schematic] FIG. 1 is a schematic diagram of the structure of the microcell of the present invention. Figure 2 is a particle size analysis of the micelles of the present invention. Figure 3 is a comparison of the interface potentials of the micelles of the present invention. Figures 4 to 6 are the acid-base response analysis of the microcells of the present invention. Figure 7 is a cytotoxicity test of the micelles of the present invention. [Main component symbol description] 1 ~ group chain copolymer; , 2 ~ gene; 3 ~ micelle.

0424-A21354TWF(N2);P02940056TW; david 18

Claims (1)

1286076 ... v * X. Patent application scope: 1. A group chain copolymer polymer, comprising A segment and B segment, wherein the A segment is polyamine and the B segment is polyethyleneimine and polyamine. Copolymer. 2. The copolymerization polymer according to claim 1, wherein the copolymerization polymer is a two-chain copolymer. 3. The group copolymerized polymer according to claim 1, wherein the B segment is positively charged in the water. 4. The copolymerization polymer according to claim 1, wherein the polyamine compound comprises poly(oxazoline, POz) or a derivative thereof. 5. The group copolymerized polymer according to claim 1, wherein the polyethyleneimine comprises a linear polyethyleneimine. 6. The group copolymerized polymer according to claim 1, wherein the polyethyleneimine comprises a branched polyethyleneimine. 7. The copolymerization polymer according to claim 1, wherein the copolymerization polymer has the following chemical formula (I): L+N - ΟΗ2-〇Η2|^+αΗ2-〇Η2-ΝΗ ^·αΗ2·αΗ2-Ν^Η_ • 4 z CD where L includes nitrogen or alkyl; Ζ includes hydrogen or C 2-21 broth; D includes sulphur-sulfur, sulfhydryl or ester group; and X, m and η It is 1 to 10,000. 8. The copolymerization polymer according to claim 7, wherein x/(m+n) is 0 to 5. 9. The copolymerization polymer of the group as described in claim 7, wherein 0424-A21354TWF(N2); P02940056TW; david 19 1286076, *r* is the initiator of ring-opening polymerization. 10. A nanocell, comprising: a plurality of agglomerate copolymers as described in claim 1; and a DNA fragment coated within the cell. 11. The nanocapsule of claim 10, wherein the weight ratio of the copolymerized polymer to the DNA fragment is substantially between 1:100 and 100:1. 12. The nanocapsule of claim 10, wherein the DNA segment and the B segment of the copolymerized polymer are bonded to each other by an electrostatic force. 13. The nanocell of the invention of claim 10, wherein the A segment of the copolymerized polymer is exposed to the outside of the microcell. 14. The nanocell of the invention of claim 10, wherein the inside of the nanocell is hydrophobic and the exterior is hydrophilic. 15. The nanocapsule of claim 10, wherein the nanocell has a particle size substantially between 10 and 1, 〇〇〇 nanometer. Φ 16. The nanocapsule of claim 10, wherein the interface potential of the nanocell is substantially between -50 and 50 mV. 17. The nanocell of the invention of claim 10, wherein the nanocell is electrically neutral. 18. The nanocell of the invention of claim 10, wherein the nanocell is disintegrated in an acidic environment to release the DNA fragment. 19. The nanocapsule of claim 18, wherein the pH of the acidic environment is substantially less than 5.5. 0424-A21354TWF(N2);P02940056TW; david 20