Synthesis and Characteristics of Biodegradable and Temperature Responsive Polymeric Micelles Based...

Accepted Manuscript

Title: Synthesis and characteristics of biodegradable andtemperature responsive polymeric micelles based onpoly(aspartic acid)-g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)

Authors: Jih-Chao Yeh, Huei-Hung Yang, Ya-Ting Hsu,Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou

PII: S0927-7757(12)00874-6DOI: doi:10.1016/j.colsurfa.2012.12.014Reference: COLSUA 18077

To appear in: Colloids and Surfaces A: Physicochem. Eng. AspectsReceived date: 13-5-2012Revised date: 11-12-2012Accepted date: 12-12-2012

Please cite this article as: Jih-Chao Yeh, Huei-Hung Yang, Ya-Ting Hsu, Chao-Ming Su, Tsong-Hai Lee, Shyh-Liang Lou,Synthesisandcharacteristicsofbiodegradableandtemperatureresponsivepolymericmicellesbasedonpoly(asparticacid)-g-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide), Colloids and SurfacesA: Physicochemical and Engineering Aspects doi:10.1016/j.colsurfa.2012.12.014This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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We synthesized PASp-g-PND possessing temperature response and

biodegradability

The micelles phase transition temperature is about 41.9oC

The micelles degraded within three days is about 25%

The micelles contains free amino groups to be able to conjugate with

bio-molecules

The micelles has potential to be used as a drug carrier for targeting treatments

*Highlights (for review)

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Synthesis and characteristics of biodegradable and temperature

responsive polymeric micelles based on poly(aspartic acid)-g-

poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)

Jih-Chao Yeha,b

, Huei-Hung Yanga, Ya-Ting Hsu

a, Chao-Ming Su

a, Tsong-Hai Lee

b,*, Shyh-

Liang Loua,**

a Department of Biomedical Engineering, Chung-Yuan Christian University, Chung Li 32023,

Taiwan, ROC

b Department of Neurology and Stroke center, Chang Gung Memorial Hospital, Linkou Medical

Center and College of Medicine, Chang Gung University, Taoyuan, Taiwan, ROC.

Both Tsong-Hai Lee and Shyh-Liang Lou act as co-corresponding authors to this manuscript.

Acknowledgment and reprint request to

Shyh-Liang Lou

Tel.: +886 3 2654517fax: +886 3 2654599.

E-mail addresses: [email protected]

200, Chung Pei Road, Chung Li 32023, Taiwan, R.O.C

*Manuscript

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Abstract

Temperature responsive polymeric micelles can release drug at controlled rate and

minimize the side effects, and have been widely used for drug delivery system. In this study,

biodegradable and temperature responsive micelles comprised of poly(aspartic acid)-g-poly(N-

isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g-poly(NIPAAm-co-DMAAm)) were

successfully synthesized by grafting poly(NIPAAm-co-DMAAm) onto poly(succinimide) and

followed by aminolysis with ammonia hydroxide. The micelles had free amine group and

exhibited a phase transition temperature above normal body temperature, which was suitable for

targeting drug delivery. In addition, PASp-g-poly(NIPAAm-co-DMAAm) was stable in

phosphate buffer solution at 37oC, and 25% of micelles could degrade within 3 days. Based on

these results, the present study demonstrated that PASp-g-poly(NIPAAm-co-DMAAm) can be

used as a drug carrier for targeting treatment.

Keywords: biodegradable, temperature responsive polymeric micelles, phase transition

temperature, drug carrier

1. Introduction

Polymeric micelles have become an interesting system for delivery of poorly water-soluble

drugs and are increasingly used in different medical applications, e.g., drug carriers for cancer

therapy[1], gene[2], protein[3] and imaging agents[4, 5]. These micelles are block or graft

copolymers that are made up of hydrophobic core and hydrophilic shell and can self-assemble in

aqueous solution by hydrophobic/hydrophilic interaction[6, 7], hydrogen bonding[8], and

electrostatic interactions[9]. Hydrophobic inner core can accommodate hydrophobic drugs, and

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hydrophilic outer shell makes particles stable in the blood stream to avoid being recognized by

reticuloendothelial system (RES) to increase treatment efficiency[10].

The stimuli-responsive micelles can sense the surrounding environment change, such as pH,

temperature, enzyme, and ion change, and response as a phase transition to control drug release.

In addition, stimuli-responsive micelles enable drugs to accumulate at the desired site either

passively by enhanced permeation and retention effect or actively through the conjugation of

recognition signal onto the surface of the micelles[11, 12]. Therefore, stimuli-responsive

polymeric micelles have been paid much attention in drug delivery system, because these

micelles can release drug at controlled rate and minimize the side effects[13].

Poly(N-isopropylacryamide), PNIPAAm, is one of the most commonly investigated

temperature responsive amphiphilic polymer, which exhibits lower critical solution temperature

(LCST) at about 32oC in aqueous media. Below the LCST, PNIPAAm is hydrophilic and

extended in the solution, which forms hydrogen bonds between the water and the amide side

chain. When above the LCST, they become water-insoluble and undergo volume transition to

aggregate in the solution. In addition, the LCST of PNIPAAm can be easily modified by

introducing hydrophilic or hydrophobic segments. Using temperature responsive micelles for

drug delivery at specific sites, the LCST of micelles needs modification to become slightly

higher than the body temperature by incorporating hydrophilic co-monomers, such as N,N-

(dimethyl amino)ethyl methacrylate[14], methyacrylic acid[15], acrylic acid[16] and

polyethylene glycol[17]. The release profile of doxorubicin in temperature responsive micelles

made from poly(N-isopropylacryamide-co-N,N-dimethylacrylamide-co-10-undecenoic acid)

with various compositions has been investigated. The micelles in phosphate buffer solution

exhibited LCST from 33 to 43oC. Also, the micelles can be stable in the blood stream at 37

oC,

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but can be deformed to release the encapsulated drugs above the LCST by breaking the

hydrophilic/hydrophobic balance of micelles[18].

Although PNIPAAm based polymeric micelles offer the advantage of temperature

responsive behavior and can act as a potential candidate for drug delivery system, they are not

biodegradable. Recently, there is methodology to improve the effectiveness of PNIPAAm based

copolymers by grafting onto biodegradable polymers. The biodegradable and temperature

responsive polymer micelles made from dextran-g-poly(NIPAAm-co-DMAAm) and chitosan-g-

poly(NIPAAm-co-DMAAm) have been studied[19-21]. The LCST of micelles can be increased

to 38oC by grafting poly(NIPAAm-co-DMAAm) onto the main chain of dextran and chitosan.

Chitosan has reactive amine groups which serve to conjugate with specific antibody for targeting

delivery. However, antidextran antibodies in human limit the cell uptake and reduce the

therapeutic effectiveness for targeting treatment.

Poly(amino acid) and its derivatives have high biocompatibility, biodegradability and

diversity in the various side chain groups for conjugating to antibodies, such as poly(aspartic

acid)[22], poly(glutamic acid)[23] and polylysine[6], which have been used for drug delivery

system. Poly(aspartic acid), PASp, is a typical poly(amino acid) derivative and is a

biodegradable, nontoxic, nonantigenic material. It can be synthesized by thermal

polycondensation of L-aspartic acid and is followed by aminolysis with amino hydroxide. The

synthesis and characteristics of amphiphilic biodegradable poly(aspartic acid-co-lactic acid) has

been investigated[24]. The degradation rate of copolymer can be increased with higher aspartic

acid content.

This study thus consists of three parts: (1) In order to increase the LCST of temperature

responsive micelles, DMAAm was used to adjust the LCST of poly(NIPAAm-co-DMAAm).

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poly(NIPAAm-co-DMAAm) with three different compositions including PN35D5 (the feed ratio

of NIPAAm and DMAAm =35:5), PN35D10 (35:10), and PN35D15 (35:15) which was

synthesized by radical copolymerization. (2) The biodegradable and temperature responsive

micelles were prepared by grafting the poly(NIPAAm-co-DMAAm) onto the main chain of

poly(aspartic acid) via aminolysis of polysuccinimide (PSI) through amino-terminated

poly(NIPAAm-co-DMAAm), followed with aminolysis of residual PSI by ammonium

hydroxide. (3) The structure analysis, LCST, biodegradable ratio in PBS and cytotoxicity of

PASp-g-poly(NIPAAm-co-DMAAm) were also discussed.

2. Materials and methods

2.1 Materials

N-Isopropylacrylamide was received from Tokyo chemical industry (TCI, Tokyo, Japan).

N,N-dimethylacrylamide and hydrazine (98%) were purchased from Alfa Aesar (MA, USA).

Methyl 3-mercaptopropionate (98%), L-aspartic acid (98%), thiazolyl blue tetrazolium bromide

(97.5%), dimethyl sulfoxide, and 1,6-diphenyl-1,3,5-hexatriene (98%) were supplied by Sigma

(MO, USA). Potassium peroxydisulfate, N,N-dimethyl methanamide (99.9%), and ammonium

hydroxide (30%) were obtained from J. T. Baker (NJ, USA). N-methylprollidone (99.9%) was

received from Showa Denko (Tokyo, Japan). Dimethyl sulfoxide-d6 (99.9%) was purchased from

Merck (Darmstadt, Germany). Orthophosphoric acid (80%) was supplied by Riedel-de Haen

(USA). Potassium bromide was obtained from Scharlan (Barcelona, Spain). Modified eagles

medium was received from GIBCO (Life Technology, NY, USA). All chemicals, reagents and

solvents were used without further purification.

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2.2 Synthesis of amino-terminated poly(N-isopropylacrylamide-co-N,N-dimethylmethanamide)

poly(NIPAAm-co-DMAAm)

Briefly, 0.41 g of NIPAAm, 0.096 mL of DMAAm and 0.02764 mL of methyl 3-mercapto

propionate were dissolved in 40 mL of deionized water and degassed with nitrogen gas for 30

min. The mixture solution was heated to 50oC under nitrogen gas. Then, 0.03 g of potassium

peroxydisulfate was dissolved in 10 mL of deionized water. The solution was added into the

monomer solution and reacted at 50oC for 2 h with magnetic stirring. Upon completion, the

resulted solution was dialyzed against deionized water using a dialysis membrane (MWCO <

1200) for 24 h and was freeze-dried. Subsequently, an appropriate amount of poly(NIPAAm-co-

DMAAm)-COOCH3 was dissolved in 40 mL of methanol solution. Hydrazine monohydrate was

dropwised into the solution and refluxed for 5 h with magnetic stirring. Upon completion, the

solution was dialyzed against deionized water using MWCO

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After 24 h, the mixture solution was cooled down to room temperature; the ammonium

hydroxide solution was added into the reaction solution and reacted at room temperature for 5 h

with stirring. The resulting solution was dialyzed against deionized water using a dialysis

membrane (MWCO

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temperature from 25 to 50oC. The LCST of the polymer was defined as the temperature

decreasing a half of absorbance of polymer solution at 50oC.

2.7 Biodegration evaluation

25 mg of PASp-g-poly(NIPAAm-co-DMAAm) was accurately weighted and dissolved in

10 mL of 0.1 M phosphate buffer saline (pH 7.4). The solution was placed in a dialysis

membrane (MWCO

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, where ODpolymer is the absorbance obtained in the presence of polymer and ODcontrol is the

absorbance obtained without polymer.

3. Results and discussion

3.1 Synthesis and characteristics of PASp-g-poly(NIPAAm-co-DMAAm)

First, PSI could be prepared by bulk polymerization of aspartic acid using phosphoric acid

as a catalyst. Then, amino-terminated poly(NIPAAm-co-DMAAm) could be synthesized by

surfactant-free radical polymerization using KPS as an initiator and methyl 3-meracptopropinate

as a chain transfer agent. Finally, the imide cyclic structure of PSI could be easily opened by an

amiable reaction. Therefore, PASp-g-poly(NIPAAm-co-DMAAm) could be prepared by

nucleophilic opening of PSI using amino-terminated poly(NIPAAm-co-DMAAm) and followed

by hydrolysis with ammonium hydroxide. The scheme of PASp-g-poly(NIPAAm-co-DMAAm)

is shown in Figure 1.

3.1.1 1H NMR analysis

The chemical structures of amino-terminated poly(NIPAAm-co-DMAAm) and PSI were

investigated by 1H NMR spectroscopy. The

1H NMR of amino-terminated poly(NIPAAm-co-

DMAAm) is shown in Figure 2(a). The characteristic peaks at 1.04 and 3.83 ppm were assigned

to the isopropyl group of NIPAAm. The peaks at 2.80 to 2.88 ppm were assigned to methyl

group on the DMAAm. In addition, the broad peak at 1.23 to 2.08 ppm was assigned to -CH2-

CH- on the polymer [25, 26]. This indicated successful copolymerization of amino-terminated

poly(NIPAAm-co-DMAAm)-HNHN2. Based on the integration of the peak area of signal a and

signal f, the actual molar ratio of NIPAAm to DMAAm could be calculated. Table 1 shows the

(2)

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actual molar ratios of NIPAAm: DMAAm were approximately equaled to the feed ratio. The 1H

NMR spectra of PSI is shown in Figure 2(b), the characteristic peaks at 2.7 to 3.21 ppm and 5.26

ppm was assigned to the protons of the methylene and methyne group on the succinimide units

of PSI [27].

The feed molar ratio of amino-terminated poly(NIPAAm-co-DMAAm) and PSI was 1:1.

The chemical structure of PASp-g-poly(NIPAAm-co-DMAAm) was analyzed by 1H NMR. As

shown in Figure 2 (c), the NMR spectra of PASp-g-poly(NIPAAm-co-DMAAm) is similar to

that of poly(NIPAAm-co-DMAAm). Upon comparison with PSI, the disappearance of the peak

at 5.26 ppm and appearance of the peak at 4.52 ppm indicated the rings in PSI were totally

opened by the amino-terminated poly(NIPAAm-co-DMAAm) and ammonium hydroxide.

According to the results of NMR analysis, the poly(NIPAAm-co-DMAAm) was successfully

grafted to the polyaspartic acid backbone chain.

3.1.2 FT-IR analysis

The FT-IR spectrum of amino-terminated poly(NIPAAm-co-DMAAm) is shown in Figure

3(a). The characteristic absorption bands at 1645 and 1550 cm-1

were assigned to the absorbance

of bending frequency of amide N-H bond, and at 3444 and 3311cm-1

were assigned to the

absorbance of N-H bond in poly(NIPAAm-co-DMAAm)[19,28,29]. The characteristic

absorption bands from the methyl 3-mercaptopropionate at 663 cm-1

was assigned to the

absorption bands of C-S bond. These results suggested there was successful polymerization of

poly(NIPAAm-co-DMAAm) and the methyl end group could be replaced by hydrazine

monohydrate.

The FT-IR spectrum of PSI is shown in Figure 3(b). The characteristic absorption bands at

1397, 1718, and 1792 cm-1

were assigned to the absorbance of methylene group, carbonyl group

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and cyclic imide of succinimide group of PSI [30,31]. Grafting of amino-terminated

poly(NIPAAm-co-DMAAm) onto the backbone of PSI followed by hydrolysis with ammonium

hydroxide was also confirmed by FT-IR spectrum of PASp-g-poly(NIPAAm-co-DMAAm)

(Figure 3(c)). The spectrum of PASp-g-poly(NIPAAm-co-DMAAm) was similar to that of

poly(NIPAAm-co-DMAAm). The characteristic peak at 1792 cm-1

assigned to the succinimide

group of PSI was disappeared. In addition, the peak at 1718 cm-1

was significantly smaller than

PSI. These results indicated there was full ring-opening of PSI by amino-terminated

poly(NIPAAm-co-DMAAm) and hydrolysis with ammonium hydroxide.

3.1.3 Molecular weight

High molecular weight and conversion of PSI was prepared by bulk polycondensation of

aspartic acid with o-phosphoric acid as a catalyst [27]. The molecular weight of PSI determined

by GPC was 30,000 Da. The conversion of aspartic acid was around 90% [32]. The LCST of

PNIPAAm was around 32oC and could be wildly used in medical applications. In addition,

DMAAm is more hydrophilic than PNIPAAm and can be used to increase the LCST of the

copolymer [17]. In this study, DMAAm is used to adjust the LCST of PNIPAAm with three

different compositions. The molecular weight of various composition poly(NIPAAm-co-

DMAAm) is summarized in Table 1. The molecular weights of PN35D5, PN35D10, and

PN35D15 determined by GPC were 7946, 10582, and 12317 Da, respectively. Using PN35D10

to conjugate with PSI, the molecular weights of PASp-g-poly(NIPAAm-co-DMAAm) was

32,130 Da. The entire polymer has good polydispersity.

3.1.4 Thermal property and miceller morphology

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The thermal weight loss curve of poly(NIPAAm-co-DMAAm), pure poly(aspartic acid),

and PASp-g-poly(NIPAAm-co-DMAAm) are shown in Figure 4. The onset of thermal

degradation of poly(NIPAAm-co-DMAAm) occurred at about 298 C and the pure poly(aspartic

acid) had multi-decomposition steps [33]. As shown in figure 4(b), PASp-g-poly(NIPAAm-co-

DMAAm) also had multi-decomposition steps. In addition, the mass loss of approximately 43%

on heating from 298 to 458 oC implied that PASp-g-poly(NIPAAm-co-DMAAm) consisted of

43% poly(NIPAAm-co-DMAAm) and 48% PASp. Based on the thermal decomposition

temperature of PASp-g-poly(NIPAAm-co-DMAAm) obtained from TGA, these micelles can be

sterilized by autoclaving.

The particle size and morphology of PASp-g-poly(NIPAAm-co-DMAAm) was observed by

DLS and TEM microscopy. TEM image of PASp-g-poly(NIPAAm-co-DMAAm) micelles is

shown in Figure 5. All PASp-g-poly(NIPAAm-co-DMAAm) micelles showed spherical shape

and the average particle size of micelles was approximately 89.1 nm. However, the particle size

analyzed by DLS was 195 nm, which was due to the dehydration of the polymer for the

measurement of TEM microscopy.

Based on the above results of FTIR, NMR, thermal property and morphology, the

biodegradable and temperature responsive polymer PASp-g-P(NIPAAm-co-DMAAm), could be

successfully synthesized and sterilized by autoclave.

3.2 Lower critical solution temperature (LCST) of polymer

The LCST of different composition of P(NIPAAm-co-DMAAm) in phosphate buffered

saline (pH 7.4) including PN35D5, PN35D10 and PN35D15 was 37.2, 40.7 and 45.7oC,

respectively (Table 1). The LCST of poly(NIPAAm-co-DMAAm) could be increased with

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increasing DMAAm monomer. In addition, the LCST of PN35D10 and PN35D15 was above

normal body temperature, which is more suitable to be used for drug releasing. After grafting of

PN35D10 onto the PSI, as shown in Figure 6, the LCST of PASp-g-PN35D10 is slightly

increased to 41.9oC. This can be explained as due to the ring-opening of PSI by PN35D10 and

ammonium hydroxide resulting in increase of hydrogen bonding between the hydrophilic

segment of PASp and water molecular, therefore, increasing the LCST. However, the LCST of

PN35D15 without grafting onto the PSI was 45.7oC, which is much higher than body

temperature. Therefore, PASp-g-PN35D10 is more suitable for drug releasing in living bodies. In

pursuance of the observations, we focused on PASp-g-PN35D10 for following investigations.

3.3 Biodegradable rate of polymer

The biodegradable rate of PASp-g-poly(NIPAAm-co-DMAAm) was studied in Phosphate

buffered saline (pH 7.4) at 37oC. As shown in Figure 7, approximately 25% of micelle weight

disappeared in the PBS within 3 days. The copolymer composition of poly(NIPAAm-co-

DMAAm)/PSI was 1. After 7 days, about 40% of micelle weight disappeared in the PBS. The

structure of degraded micelles determined by FT-IR was poly(NIPAAm-co-DMAAm). These

results suggested that the temperature responsive micelles were degraded causing chain scission

at the primary amine site in PASp-g-poly(NIPAAm-co-DMAAm). In addition, we used

PN35D10 conjugated to low molecular weight PSI (MW=10,000, poly(NIPAAAm-co-

DMAAm)/PSI=1) and investigated the biodegradable rate in PBS at 37oC (data not shown).

Approximately 40 % of micelles disappeared in the PBS within 24 h suggesting a reduced

molecular weight of PSI could be used to increase the degradable rate. Based on these research

results, it is suggested that the kidney cells can filtrate the particles with molecular weight

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smaller than 40k Da [34], which means that PASp-g-poly(NIPAAm-co-DMAAm) can be

filtrated through blood stream by kidney.

3.4 Cytotoxicity of polymer

For the ultimate of PASp-g-poly(NIPAAm-co-DMAAm) micelles as drug carrier for drug

delivery, it is important that these micelles and its degradation products retain low toxicity. L929

celles were incubated with poly(NIPAAm-co-DMAAm)(PN35D10), polyaspartic acid (PASp)

and PASp-g-poly(NIPAAm-co-DMAAm) in cultured medium containing 5mg of polymer for

24, 48, and 72 h, respectively. Figure 8 shows that the variability was above 95% and there was

no significant difference from that in the control experiment. After 72 h, PASp-g-poly(NIPAAm-

co-DMAAm) was degraded about 20% and its degradation products could be dissolved in the

cultured medium. There is no significant difference between the PASp-g-poly(NIPAAm-co-

DMAAm) and control experiment. Thus, it can be concluded that both PASp-g-poly(NIPAAm-

co-DMAAm) and its degradation products have very low or no cytotoxicity for using as drug

carriers.

4. Conclusions

The present study demonstrated that a biodegradable and temperature responsive micelle,

polyaspartic acid-g-poly(N-isopropylacryamide-co-N,N-dimethylacrylamide) (PASp-g-

poly(NIPAAm-co-DMAAm)), with a LCST at 41.6oC could be synthesized for the use as drug

delivery system. These micelles were stable in the phosphate buffered saline (pH 7.4) at 37oC,

but could be deformed and aggregated above its LCST. In addition, the micelles contained free

amine groups, which allowed further conjugation with specific antibodies for targeting treatment

and could be degraded after drug delivery. Thus, these micelles may contribute to the selective

accumulation and release of drugs in the desired site.

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Acknowledgement

The study was supported by the National Science Council, Taiwan (Contract No. NSC 98-

2314-B-182-054-MY2, NSC 99-2628-B-182-027-MY3) and Chang Gung Memorial Hospital

under the Medical Research Project (Contract No. Animal Molecular Imaging Center

CMRPG340203). And we thank Microscopy Core Laboratory of Chang Gung Memorial

Hospital at Linkou for the use of transmission electron microscopy.

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Figure 1 Synthetic scheme

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Figure 2 NMR

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Figure 3 FTIR

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Figure 5 TEM of PASPPND

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Figure 6 LCST

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Figure 7 percent degrgradation

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Figure 8 survial ratio

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