亚油酸和聚苹果酸双接枝壳聚糖新型纳米载体材料及其增强抗肿瘤药物功效研究
|
摘要
恶性肿瘤是严重威胁人类健康的重大疾病,化疗药物对肿瘤细胞的选择性差,毒副作用大,治疗效果不理想。开发新型给药系统(Drug Delivery System,DDS)提高现有抗肿瘤药物疗效,减少毒副作用,可降低新药开发的风险,药物的安全性也更加可靠,已成为癌症治疗研究的热点。纳米给药系统由于可缓、控释药物及靶向给药而受到广泛关注,纳米给药系统的载体材料必须具有良好的安全性才能进一步用于临床或生产上市。壳聚糖因良好的生物相容性和生物可降解性而获得广泛应用,是美国FDA批准的载体材料之一。本研究目的在于开发一种两亲性壳聚糖衍生物纳米粒,并对其进行PEG、叶酸(Folic acid,FA)和生物素(Biotin,BT)功能化修饰,使其成为一种安全、高效、可主动靶向肿瘤组织的抗癌药物给药载体。
以酰氯化反应制备亚油酸(LA)和聚苹果酸(PMLA)双接枝壳聚糖(LMC),希夫碱反应与EDC·HCl脱水缩合反应制备功能化修饰的LMC衍生物:PEG修饰的LMC(PEG-LMC)、叶酸修饰的PEG-LMC(FA-PEG-LMC)及生物素修饰的PEG-LMC(BT-PEG-LMC);LMC及其衍生物在水中自组装形成纳米粒;以紫杉醇(PTX)为难溶性抗肿瘤模型药物研究LMC及其衍生物纳米粒的理化性质、载药特点及体外释药规律;以H22荷瘤小鼠为模型,考察载PTX LMC及其衍生物纳米粒的抑瘤率;以SMMC-7721为肿瘤细胞模型、HEK-293为正常细胞模型研究LMC及其衍生物纳米粒的体外细胞摄取;以正常小鼠和H22荷瘤小鼠为模型,研究LMC及其衍生物纳米粒的体内组织分布及靶向性。
1 LMC及其功能化修饰衍生物的制备与表征
以D,L-天冬氨酸为起始单体,乳酸为引发剂,采用内酯开环聚合法合成一种含乳酸末端的聚苹果酸苄酯(PMLABz);以甲基磺酸为溶剂,酰氯法活化亚油酸和聚苹果酸苄酯,使其与壳聚糖的羟基以酯键连接或与氨基以酰胺键连接,制备LMC。考察亚油酸和聚苹果酸的投料比、投料顺序、反应温度及反应时间对LMC接枝反应的影响,确定LMC的制备纯化工艺:亚油酸与壳聚糖单元摩尔比为0.2～1.0,苹果酸单体与壳聚糖单元摩尔比为1.0,室温反应6 h,以甲基磺酸脱保护基,利用LMC的pH敏感性纯化产物。
DMSO-醋酸酐法制备mPEG醛,考察醋酸酐与mPEG的摩尔比、反应温度与反应时间对mPEG醛活化率的影响,制得活化率80%以上的mPEG醛。mPEG醛与LMC在DMSO和pH8.0硼酸缓冲液混合溶剂中反应,硼氢化钠还原,离心纯化制得PEG-LMC。
经EDC·HC1介导的缩合反应将叶酸和生物素接枝至PEG-LMC上,制得FA-PEG-LMC和BT-PEG-LMC。确定其制备工艺:以EDC-HC1、NHS、DMAP催化反应,透析法纯化产物。采用FTIR、~1H NMR、x射线衍射、DSC和元素分析表征了LMC及其衍生物的结构。元素分析结果表明亚油酸的取代度(DS)介于36.1～60.5之间,PMLA的DS介于0.7-1.1之间,PEG、FA和BT的DS分别为12.4,6.0和6.1。
2 LMC及其衍生物纳米粒的制备、表征及载药、释药特性
以PTX为模型药物,研究LMC及功能化修饰的LMC衍生物自组装纳米粒作为难溶性抗癌药物载体的特点。超声法制备LMC及其衍生物自组装纳米粒;透射电子显微镜(TEM)和扫描电子显微镜(SEM)考察纳米粒的形态,动态激光光散射法(DLLS)测定纳米粒的粒径与Zeta电势,稳态荧光探针法测定纳米粒的临界聚集浓度(CAC);制备载PTX LMC及其衍生物纳米粒,考察亲疏水组分比例与PEG、FA和BT修饰对包封率、载药量和体外释放行为的影响。
LMC及其衍生物纳米粒在水中的平均粒径为190～350 ilm,生理条件下Zeta电势为-2～-20mV。LMC的粒径随LA取代度的增加而减小,随PMLA链长的增加而增大,且碱性条件下的粒径大于酸性条件。LMC纳米粒的Zeta电势受介质pH值影响,pH<6.0时带正电,6.07.0时带负电。PEG修饰增大LMC纳米粒的粒径,且可屏蔽纳米粒表面电荷,使其Zeta电势绝对值降低。FA和BT修饰对PEG-LMC的粒径和Zeta电势无明显影响。LMC的CAC随LA取代度的增加而减小,PEG、FA和BT修饰不明显改变CAC。LMC及其衍生物纳米粒对PTX的包封率均超过70%,最高可达90%;载药量随LA取代度的增加而增加,最高可达9.9%,PEG修饰使载药量略有降低。载PTX LMC纳米粒4 h的累积释放率约为50～60%,8 h的累积释放率约为70-80%,可持续释放药物达24 h。LA取代度较大、PMLA链较长的LMC纳米粒释药速度较慢,PEG修饰可增强LMC纳米粒的缓释能力。
3载紫杉醇纳米粒的抗肿瘤作用、体内外靶向性及安全性
考察载PTX LMC及其衍生物纳米粒对H22荷瘤小鼠的抑瘤效果,结果抑瘤率强弱顺序为:FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX溶液,其中FA-PEG-LMC与BT-PEG-LMC的抑瘤率分别为82.5%和80.6%,可有效抑制H22肿瘤的生长。
细胞摄取试验考察LMC及其衍生物纳米粒的体外肿瘤细胞靶向性。制备罗丹明B标记的LMC纳米粒(RB-LMC NP)、PEG-LMC纳米粒(RB-PEG-LMCNP)、叶酸修饰的LMC纳米粒(RB-FA-LMC NP)、叶酸修饰的PEG-LMC纳米粒(RB-FA-PEG-LMC NP)、生物素修饰的LMC纳米粒(RB-BT-LMC NP)和生物素修饰的PEG-LMC纳米粒(RB-BT-PEG-LMC NP),考察其在SMMC-7721细胞和HEK-293细胞中的摄取。结果表明SMMC-7721肿瘤细胞对LMC及其衍生物纳米粒的摄取能力强于HEK-293正常细胞;纳米粒浓度低于1000μg·mL~(-1)时,SMMC-7721细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;纳米粒浓度低于500μg·mL~(-1)时,HEK-293细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;4 h内,SMMC-7721和HEK-293细胞对纳米粒的摄取量随共培育时间的延长而增加。FA和BT修饰的LMC纳米粒在SMMC-7721细胞中的摄取量显著高于LMC纳米粒(FA-LMC和B~LMC的摄取量分别为LMC的3～5倍和2～3倍),在HEK-293正常细胞中的摄取量显著低于SMMC-7721肿瘤细胞,表明FA和BT修饰的LMC纳米粒可选择性地主动靶向肿瘤细胞。PEG修饰对LMC纳米粒的摄取无明显影响,但同时修饰配体和PEG的FA-PEG-LMC和BT-PEG-LMC的细胞摄取量低于FA-LMC和BT-LMC。游离叶酸及生物素可抑制相应FA和BT修饰的纳米粒的摄取,对FA-PEG-LMC和BT-PEG-LMC纳米粒的抑制率高于FA-LMC和B%LMC,表明FA-LMC和BT-LMC对细胞表面叶酸和生物素受体亲和力更强。
以正常小鼠和荷H22肿瘤小鼠为模型,考察载PTX的LMC纳米粒、PEG-LMC纳米粒、FA-PEG-LMC纳米粒与BT-PEG-LMC纳米粒的体内组织分布和肿瘤靶向性。HPLC测定体内药物浓度,方法专属性、精密度、回收率均较好。PEG-LMC组血液中药物浓度时间曲线下面积(AUC)为LMC组的3.05倍,肝中AUC为LMC组的61%,脾中AUC与LMC组相近,表明PEG修饰可显著延长纳米粒在血液中的循环时间,减少肝巨噬细胞的吞噬,但无法避免脾巨噬细胞的吞噬;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC纳米粒均具有一定的肿瘤靶向性,肿瘤相对摄取率(R_e)分别为1.36、2.51、3.80、2.95,表明FA-PEG-LMC纳米粒的肿瘤靶向性最佳;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC在心、肾中的药物分布显著低于PTX溶液组,可降低PTX对心、肾的毒性。
溶血试验和急性毒性试验考察LMC纳米粒的安全性,结果LMC的溶血率低于5%;小鼠对LMC的最大耐受剂量为1250 mg·kg~(-1),耐受倍数为125倍,表明LMC作为注射给药载体安全性较高。
In the case of cancer therapy, chemotherapy is often impotent in effective treatment due to unfavorable specificity towards tumors and great side effects. Therefore, development of novel drug delivery systems (DDS) which are characterized by improved anti-tumor effect, reduced side effects, decreased cost during R&D of new drugs, and favorable safety has around great attention. Among them nanoparticles delivery systems show distinct beneficial attributes, including controlled and sustained drug release and targeted drug delivery. It is a prerequisite that the polymeric nanoparticles subjected to clinical investigations and marketing should possess desired biocompatibility and safety. Chitosan shows wide applications due to its favorable biocompatibility and biodegradability, and is an approved excipient by FDA. The current investigation aims at developing a novel kind of amphiphilic chitosan derivative nanoparticles, which are further modified with PEG, folic acid (FA) and biotin (BT), thus achieving an effective, safe, and actively targeted delivery carrier for anti-tumor drugs.
Linoleic acid (LA) and poly(malic acid) double-grafted chitosan (LMC) was prepared through acrylation reaction. Functionally modified LMC derivatives including PEG modified LMC (PEG-LMC), FA modified PEG-LMC (FA-PEG-LMC), and BT modified PEG-LMC (BT-PEG-LMC) were synthesized through Schiff base reaction and EDC·HCl mediated covalent bonding. The amphiphilic LMC and LMC derivatives self-assembled into nanoparticles in water. PTX as a model insoluble anticancer drug was loaded in LMC and LMC derivatives nanoparticles, which were characterized by drug loading efficiency, loading capacity, and in vitro release. In vitro cellular uptake of LMC and LMC derivatives nanoparticles were evaluated in SMMC-7721 cells and HEK-293 cells, and the tumor inhibition effect, biodistribution, and targeted effect were monitored in H22 bearing mice.
1 Preparation and characterization of LMC and LMC derivatives
PMLABz was synthesized through ring-open polymerization of lactone with D,L-aspartic acid as monomer and lactic acid as initiator. LA and PMLABz were activated using the acrylation method in MeSO_3H, and were conjugated with hydroxyl and amino groups on chitosan through ester and amide bonding, respectively, thus achieving the LMC. Effect of feed ratio and feed sequence of LA/poly(malic acid), reaction temperature, and reaction time on the graft reaction was assessed. The optimal molar ratio of LA/saccharide ring was set to be 0.2-1.0, molar ratio of malic acid/saccharide ring to be 1.0, reaction time to be 6 h at room temperature, and MeSO_3H was used for hydrolysis of benzyl groups.
mPEG aldehyde with an activation degree of above 80% was synthesized using the DMSO-acetic anhydride method, and the effect of acetic anhydride/mPEG molar ratio, reaction temperature and reaction time on the activation degree was evaluated. Optimally, mPEG aldehyde was allowed to conjugate with LMC in a mixed solution of DMSO and pH 8.0 borate suffer, which was deoxidized with NaBH_4 and purified through centrifugation.
Folic acid and biotin were separately conjugated to PEG-LMC via amide bond mediated by EDC·HCl, achieving the FA-PEG-LMC and BT-PEG-LMC.
FTIR, ~1H NMR, XRD, DSC, and elemental analysis confirmed synthesis of LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC. Elemental analysis demonstrated that the degree of substitution (DS) of LA was 36.1-60.5, DS of PMLA was 0.7-1.1, DS of PEG was 12.4, DS of FA was 6.0, and DS of BT was 6.1.
2 Preparation, characterization of blank and PTX-loaded LMC and LMC derivatives nanoparticles
LMC and LMC derivatives self-assembled nanoparticles with different LA and PMLA substitution degrees were prepared by sonication. Morphology of the nanoparticles was observed using TEM and SEM, and particle size and Zeta potential were monitored using DLLS. CAC was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. PTX as a model anticancer drug was loaded in the nanoparticles, and the effect of nanoparticle composition and modification of PEG, FA, and BT on encapsulation efficiency, loading capacity and in vitro release behavior was investigated.
Average particle size of LMC and its derivatives nanoparticles at pH 7.4 was 190-350 nm, and Zeta potential was -2～-20 mV. An increase in DS of LA or decrease in PMLA chain length led to decreased particle size of LMC, and particle size in alkaline conditions was larger than in acidic conditions. Zeta potential of the LMC nanoparticles was sensitive towards pH values in that they were positively charged at pH lower than 6.0, nearly uncharged within the pH range of 6.0-7.0, and negatively charged at pH higher than 7.0. The modification of PEG led to an increase in particle size and a decrease in the absolute value of Zeta potential. The modification of FA or BT had no significant effect on the particle size and Zeta potential of PEG-LMC NP. The CAC of LMC was deceased as the DS of LA enhanced and the modification of PEG, FA, and BT didn't increase CAC. Loading efficiency of LMC and its derivatives nanoparticles for PTX was above 70%, and the loading capacity increased with LA substitution degree which reached maximal value of 9.9%. The modification of PEG slightly decreased the loading capacity of LMC. A sustained release of PTX was achieved within 24 h, and an increase in LA substitution degree and PMLA chain length could slow down the release rate of PTX. The modification of PEG could enhance the slow-release effect of LMC.
3 Anti-tumor effect, tumor-targeting and safety assessment of LMC and LMC derivatives nanoparticles
Anti-tumor effect of PTX loaded LMC and LMC derivatives nanoparticles were investigated in H22 bearing mice. Both unmodified and modified PTX loaded LMC nanoparticles showed significant anti-tumor effect, while PEG modification and ligand modification resulted in improved anti-tumor effect. The tumor inhibition rate (TIR) was: FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX solution, with TIR of 82.5% and 80.6% for FA-PEG-LMC and BT-PEG-LMC, respectively.
Cellular uptake study of LMC and LMC derivatives nanoparticles were performed in SMMC-7721 cells and HEK-293 cells to evaluate the in vitro tumor targeting effect. Rhodamine B labeled LMC (RB-LMC), PEG-LMC (RB-PEG-LMC), FA-LMC (RB-FA-LMC), FA-PEG-LMC (RB-FA-PEG-LMC), BT-LMC (RB-BT-LMC), and BT-PEG-LMC (RB-BT-PEG-LMC) nanoparticles were used for assessment. The uptake of LMC and its derivatives nanoparticles in SMMC-7721 cells was much higher than that in HEK-293 cells. Cellular uptake of SMMC-7721 increased with nanoparticle concentration within 1000μg·mL~(-1), and cellular uptake of HEK-293 increased with nanoparticle concentration within 500μg·mL~(-1). Cellular uptake of SMMC-7721 and HEK-293 increased with incubation time in within 4 h. The uptake amount of FA-LMC and BT-LMC nanoparticles was 3-5 fold and 2-3 fold of that of LMC nanoparticls and the uptake amount of FA and BT modified LMC nanoparticles in HEK-293 was significantly lower than that of SMMC-7721. It meant that FA and BT modified LMC nanoparticles could actively target to tumor cells. The modification of PEG had no obviously effect on the uptake of LMC nanoparticles, but the uptake amount of FA-PEG-LMC and BT-PEG-LMC was lower than that of FA-LMC and BT-LMC. Free folic acid and biotin inhibited uptake of FA and BT modified LMC nanoparticles, respectively. The inhibition on FA-PEG-LMC and BT-PEG-LMC was stronger than that on FA-LMC and BT-LMC, which meant that FA-LMC and BT-LMC had higher affinity with FA and BT receptors on cell surface.
Biodistribution of PTX loaded LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC nanoparticles were investigated in normal and H22 bearing mice. HPLC was used for PTX quantification, and the method was precise and reliable. PEG modification prolonged retention of LMC nanoparticles in the blood circulation and decreased phagocytosis by macrophages in the liver. However, phagocytotis by the spleen was not prevented. Both unmodified and modified LMC nanoparticles showed tumor-targeting effect, while FA and BT modification yielded an additional active targeting towards tumor with FA-PEG-LMC showing the maximal tumor-target effect of R_e of 3.8. Besides, distribution of LMC and LMC derivatives nanoparticles in the heart and spleen was remarkably lower than PTX injection, indicating that toxicity of PTX towards heart and spleen was successfully reduced following encapsulation in LMC and LMC derivatives nanoparticles.
Finally, safety assessment was carried out on LMC nanoparticles in terms of hemolysis and acute toxicity. Hemolysis ratio of LMC was below 5%, and the maximum tolerance amount of LMC in mice was 1250 mg·kg~(-1), which suggested it a safety drug carrier for intravenous injection.
以酰氯化反应制备亚油酸(LA)和聚苹果酸(PMLA)双接枝壳聚糖(LMC),希夫碱反应与EDC·HCl脱水缩合反应制备功能化修饰的LMC衍生物:PEG修饰的LMC(PEG-LMC)、叶酸修饰的PEG-LMC(FA-PEG-LMC)及生物素修饰的PEG-LMC(BT-PEG-LMC);LMC及其衍生物在水中自组装形成纳米粒;以紫杉醇(PTX)为难溶性抗肿瘤模型药物研究LMC及其衍生物纳米粒的理化性质、载药特点及体外释药规律;以H22荷瘤小鼠为模型,考察载PTX LMC及其衍生物纳米粒的抑瘤率;以SMMC-7721为肿瘤细胞模型、HEK-293为正常细胞模型研究LMC及其衍生物纳米粒的体外细胞摄取;以正常小鼠和H22荷瘤小鼠为模型,研究LMC及其衍生物纳米粒的体内组织分布及靶向性。
1 LMC及其功能化修饰衍生物的制备与表征
以D,L-天冬氨酸为起始单体,乳酸为引发剂,采用内酯开环聚合法合成一种含乳酸末端的聚苹果酸苄酯(PMLABz);以甲基磺酸为溶剂,酰氯法活化亚油酸和聚苹果酸苄酯,使其与壳聚糖的羟基以酯键连接或与氨基以酰胺键连接,制备LMC。考察亚油酸和聚苹果酸的投料比、投料顺序、反应温度及反应时间对LMC接枝反应的影响,确定LMC的制备纯化工艺:亚油酸与壳聚糖单元摩尔比为0.2～1.0,苹果酸单体与壳聚糖单元摩尔比为1.0,室温反应6 h,以甲基磺酸脱保护基,利用LMC的pH敏感性纯化产物。
DMSO-醋酸酐法制备mPEG醛,考察醋酸酐与mPEG的摩尔比、反应温度与反应时间对mPEG醛活化率的影响,制得活化率80%以上的mPEG醛。mPEG醛与LMC在DMSO和pH8.0硼酸缓冲液混合溶剂中反应,硼氢化钠还原,离心纯化制得PEG-LMC。
经EDC·HC1介导的缩合反应将叶酸和生物素接枝至PEG-LMC上,制得FA-PEG-LMC和BT-PEG-LMC。确定其制备工艺:以EDC-HC1、NHS、DMAP催化反应,透析法纯化产物。采用FTIR、~1H NMR、x射线衍射、DSC和元素分析表征了LMC及其衍生物的结构。元素分析结果表明亚油酸的取代度(DS)介于36.1～60.5之间,PMLA的DS介于0.7-1.1之间,PEG、FA和BT的DS分别为12.4,6.0和6.1。
2 LMC及其衍生物纳米粒的制备、表征及载药、释药特性
以PTX为模型药物,研究LMC及功能化修饰的LMC衍生物自组装纳米粒作为难溶性抗癌药物载体的特点。超声法制备LMC及其衍生物自组装纳米粒;透射电子显微镜(TEM)和扫描电子显微镜(SEM)考察纳米粒的形态,动态激光光散射法(DLLS)测定纳米粒的粒径与Zeta电势,稳态荧光探针法测定纳米粒的临界聚集浓度(CAC);制备载PTX LMC及其衍生物纳米粒,考察亲疏水组分比例与PEG、FA和BT修饰对包封率、载药量和体外释放行为的影响。
LMC及其衍生物纳米粒在水中的平均粒径为190～350 ilm,生理条件下Zeta电势为-2～-20mV。LMC的粒径随LA取代度的增加而减小,随PMLA链长的增加而增大,且碱性条件下的粒径大于酸性条件。LMC纳米粒的Zeta电势受介质pH值影响,pH<6.0时带正电,6.0
3载紫杉醇纳米粒的抗肿瘤作用、体内外靶向性及安全性
考察载PTX LMC及其衍生物纳米粒对H22荷瘤小鼠的抑瘤效果,结果抑瘤率强弱顺序为:FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX溶液,其中FA-PEG-LMC与BT-PEG-LMC的抑瘤率分别为82.5%和80.6%,可有效抑制H22肿瘤的生长。
细胞摄取试验考察LMC及其衍生物纳米粒的体外肿瘤细胞靶向性。制备罗丹明B标记的LMC纳米粒(RB-LMC NP)、PEG-LMC纳米粒(RB-PEG-LMCNP)、叶酸修饰的LMC纳米粒(RB-FA-LMC NP)、叶酸修饰的PEG-LMC纳米粒(RB-FA-PEG-LMC NP)、生物素修饰的LMC纳米粒(RB-BT-LMC NP)和生物素修饰的PEG-LMC纳米粒(RB-BT-PEG-LMC NP),考察其在SMMC-7721细胞和HEK-293细胞中的摄取。结果表明SMMC-7721肿瘤细胞对LMC及其衍生物纳米粒的摄取能力强于HEK-293正常细胞;纳米粒浓度低于1000μg·mL~(-1)时,SMMC-7721细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;纳米粒浓度低于500μg·mL~(-1)时,HEK-293细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;4 h内,SMMC-7721和HEK-293细胞对纳米粒的摄取量随共培育时间的延长而增加。FA和BT修饰的LMC纳米粒在SMMC-7721细胞中的摄取量显著高于LMC纳米粒(FA-LMC和B~LMC的摄取量分别为LMC的3～5倍和2～3倍),在HEK-293正常细胞中的摄取量显著低于SMMC-7721肿瘤细胞,表明FA和BT修饰的LMC纳米粒可选择性地主动靶向肿瘤细胞。PEG修饰对LMC纳米粒的摄取无明显影响,但同时修饰配体和PEG的FA-PEG-LMC和BT-PEG-LMC的细胞摄取量低于FA-LMC和BT-LMC。游离叶酸及生物素可抑制相应FA和BT修饰的纳米粒的摄取,对FA-PEG-LMC和BT-PEG-LMC纳米粒的抑制率高于FA-LMC和B%LMC,表明FA-LMC和BT-LMC对细胞表面叶酸和生物素受体亲和力更强。
以正常小鼠和荷H22肿瘤小鼠为模型,考察载PTX的LMC纳米粒、PEG-LMC纳米粒、FA-PEG-LMC纳米粒与BT-PEG-LMC纳米粒的体内组织分布和肿瘤靶向性。HPLC测定体内药物浓度,方法专属性、精密度、回收率均较好。PEG-LMC组血液中药物浓度时间曲线下面积(AUC)为LMC组的3.05倍,肝中AUC为LMC组的61%,脾中AUC与LMC组相近,表明PEG修饰可显著延长纳米粒在血液中的循环时间,减少肝巨噬细胞的吞噬,但无法避免脾巨噬细胞的吞噬;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC纳米粒均具有一定的肿瘤靶向性,肿瘤相对摄取率(R_e)分别为1.36、2.51、3.80、2.95,表明FA-PEG-LMC纳米粒的肿瘤靶向性最佳;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC在心、肾中的药物分布显著低于PTX溶液组,可降低PTX对心、肾的毒性。
溶血试验和急性毒性试验考察LMC纳米粒的安全性,结果LMC的溶血率低于5%;小鼠对LMC的最大耐受剂量为1250 mg·kg~(-1),耐受倍数为125倍,表明LMC作为注射给药载体安全性较高。
In the case of cancer therapy, chemotherapy is often impotent in effective treatment due to unfavorable specificity towards tumors and great side effects. Therefore, development of novel drug delivery systems (DDS) which are characterized by improved anti-tumor effect, reduced side effects, decreased cost during R&D of new drugs, and favorable safety has around great attention. Among them nanoparticles delivery systems show distinct beneficial attributes, including controlled and sustained drug release and targeted drug delivery. It is a prerequisite that the polymeric nanoparticles subjected to clinical investigations and marketing should possess desired biocompatibility and safety. Chitosan shows wide applications due to its favorable biocompatibility and biodegradability, and is an approved excipient by FDA. The current investigation aims at developing a novel kind of amphiphilic chitosan derivative nanoparticles, which are further modified with PEG, folic acid (FA) and biotin (BT), thus achieving an effective, safe, and actively targeted delivery carrier for anti-tumor drugs.
Linoleic acid (LA) and poly(malic acid) double-grafted chitosan (LMC) was prepared through acrylation reaction. Functionally modified LMC derivatives including PEG modified LMC (PEG-LMC), FA modified PEG-LMC (FA-PEG-LMC), and BT modified PEG-LMC (BT-PEG-LMC) were synthesized through Schiff base reaction and EDC·HCl mediated covalent bonding. The amphiphilic LMC and LMC derivatives self-assembled into nanoparticles in water. PTX as a model insoluble anticancer drug was loaded in LMC and LMC derivatives nanoparticles, which were characterized by drug loading efficiency, loading capacity, and in vitro release. In vitro cellular uptake of LMC and LMC derivatives nanoparticles were evaluated in SMMC-7721 cells and HEK-293 cells, and the tumor inhibition effect, biodistribution, and targeted effect were monitored in H22 bearing mice.
1 Preparation and characterization of LMC and LMC derivatives
PMLABz was synthesized through ring-open polymerization of lactone with D,L-aspartic acid as monomer and lactic acid as initiator. LA and PMLABz were activated using the acrylation method in MeSO_3H, and were conjugated with hydroxyl and amino groups on chitosan through ester and amide bonding, respectively, thus achieving the LMC. Effect of feed ratio and feed sequence of LA/poly(malic acid), reaction temperature, and reaction time on the graft reaction was assessed. The optimal molar ratio of LA/saccharide ring was set to be 0.2-1.0, molar ratio of malic acid/saccharide ring to be 1.0, reaction time to be 6 h at room temperature, and MeSO_3H was used for hydrolysis of benzyl groups.
mPEG aldehyde with an activation degree of above 80% was synthesized using the DMSO-acetic anhydride method, and the effect of acetic anhydride/mPEG molar ratio, reaction temperature and reaction time on the activation degree was evaluated. Optimally, mPEG aldehyde was allowed to conjugate with LMC in a mixed solution of DMSO and pH 8.0 borate suffer, which was deoxidized with NaBH_4 and purified through centrifugation.
Folic acid and biotin were separately conjugated to PEG-LMC via amide bond mediated by EDC·HCl, achieving the FA-PEG-LMC and BT-PEG-LMC.
FTIR, ~1H NMR, XRD, DSC, and elemental analysis confirmed synthesis of LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC. Elemental analysis demonstrated that the degree of substitution (DS) of LA was 36.1-60.5, DS of PMLA was 0.7-1.1, DS of PEG was 12.4, DS of FA was 6.0, and DS of BT was 6.1.
2 Preparation, characterization of blank and PTX-loaded LMC and LMC derivatives nanoparticles
LMC and LMC derivatives self-assembled nanoparticles with different LA and PMLA substitution degrees were prepared by sonication. Morphology of the nanoparticles was observed using TEM and SEM, and particle size and Zeta potential were monitored using DLLS. CAC was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. PTX as a model anticancer drug was loaded in the nanoparticles, and the effect of nanoparticle composition and modification of PEG, FA, and BT on encapsulation efficiency, loading capacity and in vitro release behavior was investigated.
Average particle size of LMC and its derivatives nanoparticles at pH 7.4 was 190-350 nm, and Zeta potential was -2～-20 mV. An increase in DS of LA or decrease in PMLA chain length led to decreased particle size of LMC, and particle size in alkaline conditions was larger than in acidic conditions. Zeta potential of the LMC nanoparticles was sensitive towards pH values in that they were positively charged at pH lower than 6.0, nearly uncharged within the pH range of 6.0-7.0, and negatively charged at pH higher than 7.0. The modification of PEG led to an increase in particle size and a decrease in the absolute value of Zeta potential. The modification of FA or BT had no significant effect on the particle size and Zeta potential of PEG-LMC NP. The CAC of LMC was deceased as the DS of LA enhanced and the modification of PEG, FA, and BT didn't increase CAC. Loading efficiency of LMC and its derivatives nanoparticles for PTX was above 70%, and the loading capacity increased with LA substitution degree which reached maximal value of 9.9%. The modification of PEG slightly decreased the loading capacity of LMC. A sustained release of PTX was achieved within 24 h, and an increase in LA substitution degree and PMLA chain length could slow down the release rate of PTX. The modification of PEG could enhance the slow-release effect of LMC.
3 Anti-tumor effect, tumor-targeting and safety assessment of LMC and LMC derivatives nanoparticles
Anti-tumor effect of PTX loaded LMC and LMC derivatives nanoparticles were investigated in H22 bearing mice. Both unmodified and modified PTX loaded LMC nanoparticles showed significant anti-tumor effect, while PEG modification and ligand modification resulted in improved anti-tumor effect. The tumor inhibition rate (TIR) was: FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX solution, with TIR of 82.5% and 80.6% for FA-PEG-LMC and BT-PEG-LMC, respectively.
Cellular uptake study of LMC and LMC derivatives nanoparticles were performed in SMMC-7721 cells and HEK-293 cells to evaluate the in vitro tumor targeting effect. Rhodamine B labeled LMC (RB-LMC), PEG-LMC (RB-PEG-LMC), FA-LMC (RB-FA-LMC), FA-PEG-LMC (RB-FA-PEG-LMC), BT-LMC (RB-BT-LMC), and BT-PEG-LMC (RB-BT-PEG-LMC) nanoparticles were used for assessment. The uptake of LMC and its derivatives nanoparticles in SMMC-7721 cells was much higher than that in HEK-293 cells. Cellular uptake of SMMC-7721 increased with nanoparticle concentration within 1000μg·mL~(-1), and cellular uptake of HEK-293 increased with nanoparticle concentration within 500μg·mL~(-1). Cellular uptake of SMMC-7721 and HEK-293 increased with incubation time in within 4 h. The uptake amount of FA-LMC and BT-LMC nanoparticles was 3-5 fold and 2-3 fold of that of LMC nanoparticls and the uptake amount of FA and BT modified LMC nanoparticles in HEK-293 was significantly lower than that of SMMC-7721. It meant that FA and BT modified LMC nanoparticles could actively target to tumor cells. The modification of PEG had no obviously effect on the uptake of LMC nanoparticles, but the uptake amount of FA-PEG-LMC and BT-PEG-LMC was lower than that of FA-LMC and BT-LMC. Free folic acid and biotin inhibited uptake of FA and BT modified LMC nanoparticles, respectively. The inhibition on FA-PEG-LMC and BT-PEG-LMC was stronger than that on FA-LMC and BT-LMC, which meant that FA-LMC and BT-LMC had higher affinity with FA and BT receptors on cell surface.
Biodistribution of PTX loaded LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC nanoparticles were investigated in normal and H22 bearing mice. HPLC was used for PTX quantification, and the method was precise and reliable. PEG modification prolonged retention of LMC nanoparticles in the blood circulation and decreased phagocytosis by macrophages in the liver. However, phagocytotis by the spleen was not prevented. Both unmodified and modified LMC nanoparticles showed tumor-targeting effect, while FA and BT modification yielded an additional active targeting towards tumor with FA-PEG-LMC showing the maximal tumor-target effect of R_e of 3.8. Besides, distribution of LMC and LMC derivatives nanoparticles in the heart and spleen was remarkably lower than PTX injection, indicating that toxicity of PTX towards heart and spleen was successfully reduced following encapsulation in LMC and LMC derivatives nanoparticles.
Finally, safety assessment was carried out on LMC nanoparticles in terms of hemolysis and acute toxicity. Hemolysis ratio of LMC was below 5%, and the maximum tolerance amount of LMC in mice was 1250 mg·kg~(-1), which suggested it a safety drug carrier for intravenous injection.
引文
[1]Feng SS.Nanoparticles of biodegradable polymers for new-concept chemotherapy [J].Expert Rev Med Devic,2004,1(1):115-125.
[2]Brigger I,Dubernet C,Couvreur P.Nanoparticles in cancer therapy and diagnosis [J].Adv Drug Deliv Rev,2002,54(5):631-651.
[3]Hirsjarvi S,Peltonen L,Hirvonen J.Surface pressure measurements in particle interaction and stability studies of poly(lactic acid) nanoparticles[J].Int J Pharm,2008,348(1-2):153-160.
[4]Cheng JJ,Teply AB,Sheriff I,Sung J,Luther G,Gu FX,Levy-Nissenbaum E,Radovic-Moreno AF,Langer R,Farokhzad OC,Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery[J].Biomaterials,2007,28(5):869-876.
[5]Chen WQ,Wei H,Li SL,Feng J,Nie J,Zhang XZ,Zhuo RZ.Fabrication of star-shaped,thermo-sensitive poly(N-isopropylacrylamide)-cholic acid-poly(ε-capro -lactone) copolymers and their self-assembled micelles as drug carriers[J].Polymer,2008,49(18):3965-3972.
[6]Akagi T,Kaneko T,Kida T,Akashi M.Preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with L-phenylalanine as a protein carrier[J].J Control Release,2005,108(2-3):226-236.
[7]Chen W,Chen HR,Hu JH,Yang WL,Wang CC.Synthesis and characterization of polyion complex micelles between poly(ethylene glycol)-grafted poly(aspartic acid)and cetyltrimethyl ammonium bromide[J].Colloid Surf A,2006,278(1-3):60-66.
[8]Kommareddy S,Amiji M.Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery[J].Nanomedicine,2007,3(1):32-42.
[9]Michael JH,Patrick SS,Neil D.Protein nanoparticles as drug carriers in clinical medicine[J].Adv Drug Deliv Rev,2008,60(8):876-885.
[10]Kisich KO,Gelperina S,Higgins MP,Wilson S,Shipulo E,Oganesyan E,Heifets H.Encapsulation of moxifloxacin within poly(butyl cyanoacrylate) nanoparticles enhances efficacy against intracellular mycobacterium tuberculosis[J].Int J Pharm,2007,345(1-2):154-162.
[11] Gan Q, Wang T. Chitosan nanoparticle as protein delivery carrier-Systematic examination of fabrication conditions for efficient loading and release [J]. Colloid SurfB, 2007, 59(1): 24-34.
[12] Mitra S, Gaur U, Ghosh PC. Tumor targeted delivery of encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles as carrier [J]. J Control Release, 2001, 74(1-3): 317-323.
[13] Lin YH, Chung CK, Chen CT, Liang HF, Chen SC, Sung HW. Preparation of nanoparticles composed of chitosan/poly-gamma-glutamic acid and evaluation of their permeability through Caco-2 cells [J]. Biomacromolecules, 2005, 6(2):1104-1112.
[14] Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S. Shell cross-linked stearic acid grafted chitosan oligosacchande self-aggregated micelles for controlled release of paclitaxel [J]. Colloid Surf B, 2006, 50(2): 97-103.
[15] Sajeesh S, Sharma CP. Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery[J]. J Biomed Mater Res B, 2006, 76B: 298-305.
[16] Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW. Acetyl Histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release [J]. J Control Release, 2006, 115(1): 37-45.
[17] Braud C, Vert M. Degradation of poly((3-malic acid)-monitoring of oligomers formation by aqueous SEC and HPCE [J]. Polym Bull, 1992, 29(1-2): 177-183.
[18] Osanai S, Nakamura K. Effects of complexation between liposome and poly(malic acid) on aggregation and leakage behaviour [J]. Biomaterials, 2000, 21(9):867-876.
[19] Cammas-Marion S, Bear MM, Harada A, Guerin P, Kataoka K. New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester [J]. Macromol Chem Phys, 2000, 201(3): 355-364.
[20] Lee BS, Fujita M, Khazenzon NM, Wawrowsky KA, Wachsmann-Hogiu S, Farkas DL, Black KL, Ljubimova JY, Holler E. Polycefin, a new prototype of a multifunctional nonconjugate based on poly(beta-L-malic acid) for drug delivery [J].Bioconjugate Chem, 2006, 17(2): 317-326.
[21] Otsuka H, Nagasaki Y, Kataoka K. PEGylated nanoparticles for biological and pharmaceutical applications [J]. Adv Drug Deliv Rev, 2003, 55(3): 403-419.
[22]Hu Y,Jiang XQ,Ding Y,Zhang LY,Yang CZ,Zhang JF,Chen JN,Yang YH.Preparation and drug release behaviors of nimodipine-loaded poly(caprolactone)-poly(ethylene oxide)-polylactide amphiphilic copolymer nanoparticles[J].Biomaterials,2003,24(13):2395-2404.
[23]Nakase I,Gallis B,Takatani-Nakase T,Oh S,Lacoste E,Singh NP,Goodlett DR,Tanaka S,Futaki S,Lai H,Sasaki T.Transferrin receptor-dependent cytotoxicity of artemisinin-transferring conjugates on prostate cancer cells and induction of apoptosis [J].Cancer Lett,2009,274(2):290-298.
[24]Oh S,Kim BJ,Singh NP,Lai H,Sasaki T.Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide[J].Cancer Lett,2009,274(1):33-39.
[25]Emonard H,Bellon G,Diesbach P,Mettlen M,Hornebeck W,Courtoy PJ.Regulation of matrix metalloproteinase(MMP) activity by the low-density lipoprotein receptor-related protein(LRP).A new function for an “old friend”[J].Biochimie,2005,87(3-4):369-376.
[26]Zhao WH,Zhang QY,Kang XM,Jin S,Lou CJ.AIBI is required for the acquisition of epithelial growth factor receptor-mediated tamoxifen resistance in breast cancer cells[J].Biochem Bioph Res Co,2009,380(3):699-704.
[27]Gabor F,Bogner E,Weissenboeck A,Wirth M.The lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery[J].Adv Drug Deliv Rev,2004,56(4):459-480:
[28]Leamon CP,Low PS.Delivery of macromolecules into living cells:a method that exploits folate receptor endocytosis[J].P Natl Acad Sci USA,1991,88(13):5572-5576.
[29]Pan J,Feng SS.Targeted delivery of paclitaxel using folate-decorated poly(lactide) evitamin E TPGS nanoparticles[J].Biomaterials,2008,29(17):2663-2672.
[30]Sakahara H,Saga T.Avidin-biotin system for delivery of diagnostic agents[J].Adv Drug Deliv Rev,1999,37(1-3):89-101.
[31]Correa-Gonzalez L,de Murphy CA,Ferro-Flores G,Pedraza-Lopez M,Murphy-Stack E,Mino-Leon D,Perez-Villasenor G,Diaz-Torres Y,Munoz-Olvera R.Uptake of ~(153)Sm-DAPA-bis-biotin and ~(99m)Tc-DTPA-bis-biotin in rat AS-30D-hepatoma cells[J].Nucl Med Biol,2003,30(2):135-140.
[32]Na K,Lee TB,Park KH,Shin EK,Lee YB,Choi HK.Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as at argeted anti-cancer drug delivery system [J]. Eur JPharm Sci, 2003, 18(2): 165-173.
[33] Negishi T, Koizumi F, Uchino F, Kuroda J, Kawaguchi T, Naito S, Matsumura Y. NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel [J]. Brit J Cancer, 2006, 95(5):601-606.
[34] Soepenberg O, Sparreboom A, Jonge MJA, Planting AST, Heus G, Loos WJ,Hartman CM, Bowden C, Verweij J. Real-time pharmacokinetics guiding clinical decisions: phase I study of a weekly schedule of liposome with solid encapsulated paclitaxel in patients tumours [J]. Eur J Cancer, 2004, 40(5): 681-688.
[35] You JO, Auguste DT. Feedback-regulated paclitaxel delivery based on poly(N,N-dimethylaminoethyl methacrylate-co-2-Hydroxyethyl methacrylate) nanoparticles [J]. Biomaterials, 2008, 29(12): 1950-1957.
[1]Service RF.Nanotechnology takes aim at cancer[J].Science,2005,310(5751):1132-1134.
[2]LaVan DA,McGuire T,Langer R.Small-scale systems for in vivo drug delivery [J].Nat Biotechnol,2003,21(10):1184-1191.
[3]LaVan DA,Lynn DM,Langer R.Moving smaller in drug discovery and delivery [J].Nat Rev Drug Discov,2002,1(1):77-84.
[4]Lesniak MS,Brem H.Targeted therapy for brain tumors[J].Nat Rev Drug Discov,2004,3(6):499-508.
[5]Chen ML.Lipid excipients and delivery systems for pharmaceutical development:A regulatory perspective[J].Adv Drug Deliv Rev,2008,60(10):768-777.
[6]蒋挺大.壳聚糖(第二版)[M].北京:化学工业出版社,2006:12.
[7]Harris JM,Struck EC,Case MG,Paley MS,Vanalstine JM,Brookes DE.Synthesis and characterization of poly(ethylene glycol) derivatives[J].J Polym Sci Pol Chem,1984,22(2):341-352.
[8]Kajiyama T,Kobayashi H,Taguchi T,Kataoka K,Tanaka J.Improved synthesis with high yield and increased molecular weight of poly(α,β-malic acid) by direct polycondensation[J].Biomacromolecules,2004,5(1):169-174.
[9]Portilla-Arias JA,Garcia-Alvarez M,de Ilarduya AM,Holler E,Munoz-Guerra S.Thermal decomposition of fungal poly(β,L-malic acid) and poly(β,L-malate)s[J].Biomacromolecules,2006,7(11):3283-3290.
[10]Vert M.Chemical routes to poly(β-malic acid) and potential applications of this water-soluble bioresorbable poly(β-hydroxy alkanoate)[J].Polym Degrad Stabil,1998,59(1-3):169-175.
[11]Coulembier O,Degee P,Hedrick JL,Dubois P.From controlled ring-opening polymerization to biodegradable aliphatic polyester Especially poly(β-malic acid)derivatives[J].Prog Polym Sci,2006,31(8):723-747.
[12]Guering P,Vert M,Braud C,Lenz RW.Optically active poly(β-malic acid)[J].Polym Bull,1985,14(2):187-192.
[13]Fukuzaki H,Yoshida M,Asano M,Kumakura M.Synthesis of copoly (D,L-lactic acid) with relatively low-molecular weight and in vitro degradation[J].Eur Polym J,1989,25(10):1019-1026.
[14]Imasaka K,Nagai T,Yoshida M,Fukuzaki H,Asano M,Kumakura M.Direct copolycondensation of epsilon-caprolactone with delta-valerolactone in the absence of catalysts.Eur Polym J,1990,26(8):831-836.
[15]解德良,姜标,杨昌正.羟基酸引发ε-己内酯开环聚合的研究[J].高分子学报,2000,5:532-537.
[16]李玉宝,生物医学材料[M].北京:化学工业出版社,2003:211.
[17]Sashiwa H,Kawasaki N,Nakayama A,Muraki E,Yamamoto N,Zhu H,Nagano H,Omura Y,Saimoto H,Shigemasa Y,Aiba SI.Chemical modification of chitosan 13Synthesis of organosoluble,palladium adsorbable,and biodegradable chitosan derivatives toward the chemical plating on plastics[J].Biomacromolecules,2002,3(5):1120-1125.
[18]Llanos GR,Sefton MV.Immobilization of poly(ethylene glycol) onto a poly(vinyl alcohol) hydrogel[J].Macromolecules,1991,24(23):6065-6072.
[19]Samuels RJ.Solid state characterization of the structure of chitosan films[J].J Polym Sci Pol Phys,1981,19(7):1081-1105.
[20]Ganji F,Abdekhodaie MJ.Synthesis and characterization of a new thermosensitive chitosan-PEG diblock copolymer[J].Carbohyd Polym,2008,74(3):435-441.
[21]Gupta KC,Jabrail FH.Effects of degree of deacetylation and cross-linking on physical characteristics,swelling and release behavior of chitosan microspheres[J].Carbohyd Polym,2006,66(1):43-54.
[1]Schmid EF,Smith DA.R&D technology investments:misguided and expensive or a better way to discover medicines?[J].Drug Discov Today,2006,11(17-18):775-784.
[2]Allen TM,Cullis PR.Drug delivery system:Entering the mainstream[J].Science,2004,303(5665):1818-1822.
[3]Duncan R.The dawning era of polymer therapeutics[J].Nat Rev Drug Discov,2003,2(5):347-360.
[4]Shiladitya S,David E,Ishan C,Zhao G,Nichi W,Tanyel K,Ram S.Temporal targeting of tumor cells and neovasculature with a nanoscale delivery system[J].Nature,2005,436(7050):568-572.
[5]Miwa A,Ishibe A,Nakano M,Yamahira T,Itai S,Jinno S,Kawahara H.Development of novel chitosan derivatives as micellar carriers of Taxol[J].Pharm Res,1998,15(12):1844-1850.
[6]Liu CG,Desai KGH,Chen XG,Park HJ.Linoleic acid-modified chitosan for formation of self-assembled nanoparticles[J].J Agr Food Chem,2005,53(2):437-441.
[7]Park JH,Kwon SG,Nam JO,Park RW,Chung H,Seo SB,Kim IS,Kwon IC,Jeong SY.Self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid for RGD peptide delivery[J].J Control Release,2004,95(3):579-588.
[8]Zhang C,Ping QN,Zhang HJ,Shen J.Preparation of N-alkyl-O-sulfate chitosan derivatives and micellar solubilization of taxol[J].Carbohyd Polym,2003,54(2):137-141.
[9]Yang XD,Zhang QQ,Wang YS,Chen H,Zhang HZ,Gao FP,Liu LR.Self-aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: Synthesis; characterization; aggregation and methotrexate release in vitro [J]. Colloid Surf B,2008,61(2): 125-131.
[10] Liu ZH, Jiao YP, Wang YF, Zhou CR, Zhang ZY. Polysaccharides-based nanoparticles as drug delivery systems [J]. Adv Drug Deliv Rev, 2008, 60(15): 1650-1662.
[11] Prabahran M, Grailer JJ, Pilla S, Steeber DA, Gong SQ. Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn~® H40, poly(1-lactide) and poly (ethylene glycol) for tumor-targeted drug delivery [J]. Biomaterials, 2009, 30 (16): 3009-3019.
[12] Seo JH, Moon HS, Kim IY, Guo DD, Lee HG, Choi YJ, Cho CS. PEGylated conjugated linoleic acid stimulation of apoptosis via a p53-mediated signaling pathway in MCF-7 breastcancer cells [J]. Eur J Pharm Biopharm, 2008, 70 (2): 621-626.
[13] Pyrz WD, Buttrey DJ. Particle size determination using TEM: A discussion of image acquisition and analysis for the novice microscopist [J]. Langmuir, 2008, 24(20):11350-11360.
[14] Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW. N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis, exocytosis and drug release [J]. J Control Release, 2006, 115 (1): 37-45.
[15] Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, Kim Y, Park JH, Kim J, Her S, Oh YK, Kwon IC, Kim K, Jeon SY. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles [J]. J Control Release, 2009, 13 (3): 259-267.
[16] Li FQ, Su H, Wang J, Liu JY, Zhu QG, Fei YB, Pan YH, Hu JH. Preparation and characterization of sodium ferulate entrapped bovine serum albumin nanoparticles for liver targeting [J]. Int J Pharm, 2008, 349 (1-2): 274-282.
[17] Li SD, Huang L. Pharmacokinetics and biodistribution of nanoparticles [J]. Mol Pharm, 2008, 5 (4): 496-504.
[18] Dobrovolskaia MA, Aggarwal P, Hall JB, Mcneil SE. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution [J]. Mol Pharm, 2008, 5 (4): 487-495.
[19] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles [J]. Mol Pharm, 2008, 5 (4): 505-515.
[20] Manjunath K, Venkateswarlu. Pharmacokinetics, tissue distribution and bioavailability of clozapine solid lipid nanoparticles after intravenous and intraduodenal administration [J]. J Control Release, 2005, 107 (2): 215-228.
[21] Dutta P, Dey J, Ghosh G, Nayak RR. Self-association and microenvironment of random
amphiphilic copolymers of sodium N-acryloyl-L-valinate and N-dodecylacrylamide in
aqueous solution [J]. Polymer, 2009, 50 (6): 1516-1525.
[22] Deepa S, Mishra AK. Fluorescence spectroscopic study of serum albumin-bromadiolone
interaction: fluorimetric determination of bromadiolone [J]. J Pharmaceut Biomed, 2005, 38
(3): 556-563.
[23] Fuguet E, Rafols C, Roses M, Bosch E. Critical micelle concentration of surfactants in
aqueous buffered and unbuffered systems [J]. Anal Chim Acta, 2005, 548(1-2): 95-100.
[24] Srivastava A, Waite JH, Stucky GD, Mikhailovsky A. Fluorescence investigations into
complex coacervation between polyvinylimidazole and sodium alginate [J]. Macromolecules,
2009, 42 (6): 2168-2176.
[25] Tan YL, Liu CG. Self-aggregated nanoparticles from linoleic acid modified
carboxymethyl chitosan: Synthesis, characterization and application in vitro [J]. Colloid Surf
B, 2009, 69 (2): 178-182.
[26] Danhier F, Lecouturier N, Vroman B, Jerome C, Marchand-brynaert J, Feron O, Preat V.
Paclitaxel-loaded PEGylated PLGA-based nanoparticles: In vitro and in vivo evaluation [J]. J
Control Release, 2009, 133 (1): 11-17.
[27] Jung J, Lee IH, Lee E, Park J, Jon S. pH-Sensitive polymer nanospheres for use as a
potential drug delivery vehicle [J]. Biomacromolecules, 2007, 8 (11): 3401-3407.
[28] Xu ZH, Chen LL, Gu WW, Gao Y, Lin LP, Zhang ZW, Zi Y, Li YP. The performance of
decetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma [J].
Biomaterials, 2009, 30 (2): 226-232.
[29] Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S. Shell cross-linked stearic acid grafted chitosan
oligosaccharide self-aggregated micelles for controlled release of paclitaxel [J]. Colloid Surf
B, 2006, 50 (2): 97-103.
[30] Zhang ZP, Feng SS. The drug encapsulation efficiency, in vitro drug release, cellular
uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol
succinate nanoparticles [J]. Biomaterials, 2006, 27 (21): 4025-4033.
[31] Prokop A, Davidson JM. Nanovehicular intracellular delivery systems [J]. J Pharm Sci,
2008, 97(9): 3518-3590.
[32] Mohamed F, Van der Walle CF. Engineering biodegradable polyester particles with
specific drug targeting and drug release properties [J]. J Pharm Sci, 2008, 97(1): 71-87.
[33] Lee SJ, Park K, Oh YK, Kwon SH, Her S, Kim IS, Choi K, Lee SJ, Kim H, Lee SG,
Kim K, Kwon IC. Tumor specificity and therapeutic efficacy of photosensitizer-encapsulated
43 glycol chitosan-based nanoparticles in tumor-bearing mice[J].Biomaterials,2009,30(15):2929-2939.
[1]Davis HE,Chen Z,Shin DM.Nanoparticle therapeutics:an emerging treatment modality for cancer[J].Nat Rev Drug Discov,2008,7(9):771-782.
[2]王晓华.生物化学与分子生物学实验技术[M].北京:化学工业出版社,2008:74-75.
[3]张忠泉,陈百泉.药理学实验指导[M].郑州:郑州大学出版社,2007:21.
[4]张馨欣,甘勇,杨星钢,朱春柳,甘莉,聂淑芳,潘卫三.聚乙二醇修饰的羟基喜树碱纳米粒脂质载体的制备及小鼠组织分布[J].药学学报,2008,43(1):91-96
[5]Williams J,Lansdown R,Sweitzer R,Romanowski M,LaBell R,Ramaswami R,Unger E.Nanoparticle drug delivery system for intravenous delivery of topoisomerase inhibitors[J].J Control Release,2003,91(1-2):167-172.
[6]Liang XJ,Shen DW,Garfield S,Gottesman MM.Mislocalization of membrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines[J].Cancer Res,2003,63(18):5909-5916.
[7]Zhang LY,Yang M,Wang Q,Li Y,Guo R,Jiang XQ,Yang CZ,Liu BR.10-Hydroxycamptothecin loaded nanoparticles:Preparation and antitumor activity in mice[J].J Control Release,2007,112(2):153-162.
[8]Mao S,Germershaus O,Fischer D,Linn T,Schnepf R,Kissel T.Uptake and transport of PEG-graft-trimethyl-chitosan copolymer-insulin nanocomplexes by epithelial cells[J].Pharm Res,2005,22(12):2058-2068.
[9]Jae HP,Seulki L,Jong-Ho K,Kyeongsoon P,Kwangmeyung K,Ick CK.Polymeric nanomedicine for cancer therapy[J].Prog Polym Sci,2008,33(1): 113-137.
[10]Nam HY,Kwon SM,Chung H,Lee SY,Kwon SH,Jeon H,Kim Y,Park JH,Kim J,Her S,Oh YK,Kwon IC,Kim K,Jeong SY.Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles[J].J Control Release,2009,135(3):259-267.
[11]Yakashi Y,Yoshiyuki H,Motoki H,Kimiko K,Yoshie M.Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts[J].Eur J Pharm Biopharm,2008,70(3):718-725.
[12]Cristina L Z,William TP,Anuradha S.Use of avidin/biotin-liposome system for enhanced peritoneal drug delivery in an ovarian cancer model[J].Int J Pharm,2007,337(1/2):316-328.
[13]Hilgenbrink AR,Low PS.Folate receptor-mediated drug targeting from therapeutics to diagnostics[J].J Pharm Sci,2005,94(10):2135-2146.
[14]Patil YB,Toti US,Khdair A,Mac L,Panyamc J.Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery[J].Biomaterials,2009,30(5):859-866.
[15]张丽珺,方晓玲.聚合物纳米粒和胶束的主动靶向研究进展[J].国外医学药学分册,2005,32(6):399-403.
[16]Kimura M,Umegaki K,Higuchi M,Thomas P,Fenech M.Methylenetetrahydrofolate reductase C677T polymorphism,folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes[J].J Nutr,2004,134(1):48-56.
[17]Mock DM,Lankford GL,Mock NI.Biotin accounts for only half of the total avidin-binding substances in human serum[J].J Nutr,1995,125(4):941-946.
[18]Gregory RJ,Kirsten MT,John ME,John R,David N.Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumors[J].J Inorg Biochem,2004,98(10):1625-1633.
[19]中国药典 2005 版二部附录 XIXB.
[20]Yamamoto Y,Nagasaki Y,Kato Y,Sugiyama Y,Kataoka K.Long-circulating poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles with modulated surface charge[J].J Control Release,2001,77(1-2):27-38.
[21]Romberg B,Hennink WE,Storm G Sheddable coatings for long-circulating nanoparticles[J].Pharm Res,2008,25(1):55-71.
[22]Abraham AM,Walubo A.The effect of surface charge on the disposition of liposome-encapsulated gentamicin to the rat liver,brain,lungs and kidneys after intraperitoneal administration[J].Int J Antimicrob Ag,2005,25(5):392-397.
[23]Yamamoto H,Kuno Y,Sugimoto S,Takeuchi H,Kawashima Y.Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions[J].J Control Release,2005,102(2):373-381.
[24]Russell-Jones G,Mc Tavish K,McEwan J,Rice J,Nowotnik D.Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours[J].J Inorg Biochem,2004,98(10):1625-1633.
[25]Maeda H,Wu J,Sawa T,Matsumura Y.Hori K.Tumor vascular permeability and the EPR effect in macromolecular therapeutics:a review[J].J Control Release,2000,65(1-2):271-284.
[26]方超,裴元英.运载抗肿瘤药物的隐形纳米粒的研究进展[J].中国药学杂志,2004,39(2):89-92.
[27]Corrocher R,Pachor ML,Bambara LM,De Sandre G.Evidence for a folic acid binding protein in human cell membrane[J].Acta Haematol,1981,66(3):202-209.
[28]McGuigan AP,Sefton MV.The influence of biomaterials on endothelial cell thrombogenicity[J].Biomaterials,2007,28(16):2547-2571.
[29]GB/T 16886.122001.医疗器械生物学评价第1部分:评价与试验[S].北京:中国标准出版社,2002.
[30]Mayer A,Vadon M,Rinner B,Novak A,Wintersteiger R,Fr(o|¨)hlich E.The role of nanoparticle size in hemocompatibility[J].Toxicol,2009,258(2-3):139-147.
[1]Hans ML,Lowman AM.Biodegradable nanoparticles for drug delivery and targeting[J].Curr Opin Solid St M,2002,6(4):319-327.
[2]Liu WG,Sun SJ,Zhang X,De YK.Self-aggregation behavior of alkylated chitosan and its effect on the release of a hydrophobic drug[J].J Biomater Sci Polymer Edn,2003,14(8):851-859.
[3]Liu CG,Desai KGH,Chen XG,Park HJ.Linolenic acid-modified chitosan for formation of self-assembled nanoparticles[J].J Agric Food Chem,2005,53(2):437-441.
[4]Zhang J,Chen XG,Li YY,Liu CS.Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin[J].Nanomedicine,2007,3(4):258-265.
[5] Jiang GB, Quan DP , Liao KR, Wang HH. Preparation of polymeric micelles
based on chitosan bearing a small amount of highly hydrophobic groups [J].
Carbohyd Polym, 2006, 66(4): 514-520.
[6] Lee KY, Jo WH, Kwon IC, Kim YH, Jeong SY. Structural determination and
interior polarity of self-aggregates prepared from deoxycholic acid-modified chitosan
in water [J]. Macromolecules, 1998, 31(2): 378-383.
[7] Park JK, Kim DG, Choi C, Jeong YL, Kim MY, Jang MK , Nah JW. Preparation
and characterization of lithocholic acid conjugated chitosan oligosaccharide
nanoparticles for hydrophobic anticancer agent carriers [J]. Polym-Korea, 2008, 32(3):
263-269.
[8] Wang YS, Liu LR, Jiang Q, Zhang QQ. Self-aggregated nanoparticles of
cholesterol-modified chitosan conjugate as a novel carrier of epirubicin [J]. Eur
Polym J, 2007, 43(1): 43-51.
[9] Natalija C, Ricardas M. Synthesis and study of water-soluble
chitosan-O-poly(ethylene glycol) graft copolymers [J]. Eur Polym J, 2004, 40(4):
685-691.
[10] Park JS, Koh YS, Bang JY , Jeong YI, Lee JJ. Antitumor effect of all-trans
retinoic acid-encapsulated nanoparticles of methoxy poly(ethylene glycol)-conjugated
chitosan against CT-26 colon carcinoma in vitro [J]. J Pharm Sci-US, 2008, 97(9):
4011-4019.
[11] Yan CY, Chen DW, Gu JW, Qin J. Nanoparticles of 5-fluorouracil (5-FU) loaded
N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization
in vitro drug release and anti-tumour activity [J]. J Pharm Pharmacol, 2006, 58(9):
1177-1181.
[12] Kim K, Kwon S, Park JH, Chung H, Jeong SY, Kwon IC, Kim IS.
Physicochemical characterizations of self-assembled nanoparticles of glycol
chitosan-deoxycholic acid conjugates [J]. Biomacromolecules, 2005, 6(2): 1154-1158.
[13] Park JH, Kwon S, Nam JO, Park RW, Chung H, Seo SB, Kim IS, Kwon IC,
Jeong SY. Self-assembled nanoparticles based on glycol chitosan bearing 5(3-cholanic
acid for RGD peptide delivery [J]. J Control Release, 2004, 95(3): 579-588.
[14] Hwang HY, Kim IS, Kwon IC , Kim YH . Tumor targetability and antitumor
effect of docetaxel-loaded hydrophobically modified glycol chitosan nanoparticles [J].
J Control Release, 2008, 128(1): 23-31.
[15] Zhang C, Ping QN, Zhang HJ, Shen J. Preparation of N-alkyl-O-sulfate chitosan
91 derivatives and micellar solubilization of taxol[J].Carbohyd Polym,2003,54(4):137-141.
[16]Qian F,Cui FY,Ding JY,Tang C,Yin CH.Chitosan graft copolymer nanoparticles for oral protein drug delivery:preparation and characterization[J].Biomacromolecules,2006,7(10):2722-2727.
[17]Sajeesh S,Sharma CP.Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery[J].J Biomed Mater Res B,2006,76B(2):298-305.
[18]Park JS,Han TH,Lee KY,Han SS,Hwang JJ,Moon DH,Kim SY,Cho YW.N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs:endocytosis,exocytosis and drug release[J].J Control Release,2006,115(1):37-45.
[19]Liu ZH,Jiao YP,Liu F,Zhang ZY.Heparin/chitosan nanoparticle carriers prepared by polyelectrolyte complexation[J].J Biomed Mater Res A,2007,83A(3):806-812.
[20]Chen Y,Mohanraj VJ,Parkin JE.Chitosan-dextran sulfate nanoparticles for delivery of an anti-angiogenesis peptide[J].Lett Pept Sci,2003,10(5-6):621-629.
[21]Bodnar M,Hartmann JF,Borbely J.Preparation and characterization of chitosan-based nanoparticles[J].Biomacromolecules,2005,6(5):2521-2527.
[22]Janes KA,Calvo P,Alonso MJ.Polysaccharide colloidal particles as delivery systems for macromolecules[J].Adv Drug Deliv Rev,2001,47(1):83-97.
[23]Gan Q,Wang T,Cochrane C,McCarron P.Modulation of surface charge,particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery[J].Colloid Surf B,2005,44(2-3):65-73.
[24]Guan XP,Quan DP,Liao KR,Wang T,Xiang P,Mai KC.Preparation and characterization of cationic chitosan-modified poly(D,L-lactide-co-glycolide)copolymer nanospheres as DNA carriers[J].J Biomater Appl,2008,22(4):353-371.
[25]李培,朱爱梅,刘庆林.沉淀聚合法制备壳聚糖磁性微球[J].化工时刊,2008,22(7):1-4.
[26]Yu JM,Li YH,Qiu LY,Jin Y.Self-aggregated nanoparticles of cholesterol-modified glycol chitosan conjugate:Preparation,characterization,and preliminary assessment as a new drug delivery carrier[J].Eur Polym J,2008,44(3):555-565.
[27]Yoo HS,Lee JE,Chung H,Kwon IC,Jeong SY.Self-assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery[J].J Control Release,2005,103(1):235-243.
[28]Qi LF,Xu ZR.In vivo antitumor activity of chitosan nanoparticles[J].Bioorg Med Chem Lett,2006,16(16):4243-4245.
[29]Bilensoy E,Sarisozen C,Esendagli G,Dogan AL,Aktas Y,Sen M,Mungan NA.Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors[J].Int J Pharm,2009,371(1-2):170-176.
[30]Lee SJ,Park K,Oh YK,Kwon SH,Her S,Kim IS,Choi K,Lee SJ,Kim H,Lee SG,Kim K,Kwon IC.Tumor specificity and therapeutic efficacy of photosensitizer-encapsulated glycol chitosan-based nanoparticles in tumor-bearing mice[J].Biomaterials,2009,30(15):2929-2939.
[31]Min KH,Park K,Kim YS,Bae SM,Lee S,Jo HG,Park RW,Kim IS,Jeong SY,Kim K,Kwon IC.Hydrophobically modified glycol chitosan nanoparticles encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy[J].J Control Release,2008,127(3):208-218.
[32]Tan YL,Liu CG.Self-aggregated nanoparticles from linoleic acid modified carboxymethyl chitosan:Synthesis,characterizaton and applicatioin in vitro[J].Colloid Surf B,2009,69(2):178-182.
[33]Zhang J,Chen XG,Liu CS,Park HJ.Investigation of polymeric amphiphilic nanoparticles as antitumor drug carriers[J].J Mater Sci-Mater M,2009,20(4):991-999.
[34]Sarmento B,Ribeiro A,Veiga F,Ferreira D,Neufeld R.Oral bioavailability of insulin contained in polysaccharide nanoparticles[J].Biomacromolecules,2007,8(10):3054-3060.
[35]Chert F,Zhang ZR,Yuan F,Qin X,Wang MT,Huang Y.In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery[J].Int J Pharm,2008,349(1-2):226-233.
[36]胡思隽,谢勇.壳聚糖纳米粒在基因治疗中的应用[J].国际病理科学与临床杂志,2006,26(2):150-152.
[37]Zhao Z,Yu SB,Wu FL,Mao ZB,Yu CL.Transfection of primary chondrocytes using chitosan-pEGFP nanoparticles[J].J Control Release,2006,112(2):223-228.
[38]Guliyeva U,Oner F,Ozsoy S,Haziroglu R.Chitosan microparticles containing plasmid DNA as potential oral gene delivery system[J].Eur J Pharm Biopharm,2006,62(1):17-25.
[39]Liu XD,Howard KA,Dong MD,Andersen MO,Rahbek UL,Johnsen MG,Hansen OC,Besenbacher F,Kjems J.The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing[J].Biomaterials,2007,28(6):1280-1288.
[40]王琳玲.壳聚糖及其衍生物抗菌性质的研究进展[J]_上海生物医学工程,2006,27(2):111-114.
[41]Motwani SK,Chopra S,Talegaonkar S,Kohli K,Ahmad FJ,Khar RK.Chitosan-sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery:Formulation,optimization and in vitro characterization[J].Eur J Pharm Biopharm,2008,68(3):513-525.
[42]Kim EM,Jeong HJ,Kim SL,Sohn MH,Nah JW,Born HS,Park IK,Cho CS.Asialoglycoprotein-receptor-targeted hepatocyte imaging using Tc-99m galactosylated chitosan[J].Nucl Med Biol,2006,33(4):529-534.
[43]Rhee K,Moon HJ,Park KS,Khang G.Evaluation of peptides tagged nanoparticle adhesion to activated endothelial cells[A].Jarm T,Kramar P,Zupanic A.11~(th) Mediteranean Conference On Medical And Biological Engineering And Computing 2007[C].Ljubljana,Slovenia,2007:835-838.
[44]Aktas Y,Yemisci M,Andrieux K,Gursoy RN,Alonso MJ,Fernandez-Megia E,Novoa-Carballal R,Quinoa E,Riguera R,Sargon MF,Celik HH,Demir AS,Hincal AA,Dalkara T,Capan Y,Couvreur P.Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26[J].Bioconjugate Chem,2005,16(6):1503-1511
[45]Jain A,Jain SK.In vitro and cell uptake studies for targeting of ligand anchored nanoparticles for colon tumors[J].Eur J Pharm Sci,2008,35(5):404-416.
[46]Fernandes JC,Wang HJ,Jreyssaty C,Benderdour M,Lavigne P,Qiu XP,Winnik FM,Zhang XL,Dai KR,Shi Q.Bone-protective effects of nonviral gene therapy with folate-chitosan DNA nanoparticle containing interleukin-1 receptor antagonist gene in rats with adjuvant-induced arthritis[J].Mol Ther,2008,16(7):1243-1251.
[47]姚倩,侯世祥,张瑄,赵钢,荀小军,游金宗.生物素化壳聚糖纳米粒的制备及其相关性质[J].药学学报,2007,42(5):557-561.
[2]Brigger I,Dubernet C,Couvreur P.Nanoparticles in cancer therapy and diagnosis [J].Adv Drug Deliv Rev,2002,54(5):631-651.
[3]Hirsjarvi S,Peltonen L,Hirvonen J.Surface pressure measurements in particle interaction and stability studies of poly(lactic acid) nanoparticles[J].Int J Pharm,2008,348(1-2):153-160.
[4]Cheng JJ,Teply AB,Sheriff I,Sung J,Luther G,Gu FX,Levy-Nissenbaum E,Radovic-Moreno AF,Langer R,Farokhzad OC,Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery[J].Biomaterials,2007,28(5):869-876.
[5]Chen WQ,Wei H,Li SL,Feng J,Nie J,Zhang XZ,Zhuo RZ.Fabrication of star-shaped,thermo-sensitive poly(N-isopropylacrylamide)-cholic acid-poly(ε-capro -lactone) copolymers and their self-assembled micelles as drug carriers[J].Polymer,2008,49(18):3965-3972.
[6]Akagi T,Kaneko T,Kida T,Akashi M.Preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with L-phenylalanine as a protein carrier[J].J Control Release,2005,108(2-3):226-236.
[7]Chen W,Chen HR,Hu JH,Yang WL,Wang CC.Synthesis and characterization of polyion complex micelles between poly(ethylene glycol)-grafted poly(aspartic acid)and cetyltrimethyl ammonium bromide[J].Colloid Surf A,2006,278(1-3):60-66.
[8]Kommareddy S,Amiji M.Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery[J].Nanomedicine,2007,3(1):32-42.
[9]Michael JH,Patrick SS,Neil D.Protein nanoparticles as drug carriers in clinical medicine[J].Adv Drug Deliv Rev,2008,60(8):876-885.
[10]Kisich KO,Gelperina S,Higgins MP,Wilson S,Shipulo E,Oganesyan E,Heifets H.Encapsulation of moxifloxacin within poly(butyl cyanoacrylate) nanoparticles enhances efficacy against intracellular mycobacterium tuberculosis[J].Int J Pharm,2007,345(1-2):154-162.
[11] Gan Q, Wang T. Chitosan nanoparticle as protein delivery carrier-Systematic examination of fabrication conditions for efficient loading and release [J]. Colloid SurfB, 2007, 59(1): 24-34.
[12] Mitra S, Gaur U, Ghosh PC. Tumor targeted delivery of encapsulated dextran-doxorubicin conjugate using chitosan nanoparticles as carrier [J]. J Control Release, 2001, 74(1-3): 317-323.
[13] Lin YH, Chung CK, Chen CT, Liang HF, Chen SC, Sung HW. Preparation of nanoparticles composed of chitosan/poly-gamma-glutamic acid and evaluation of their permeability through Caco-2 cells [J]. Biomacromolecules, 2005, 6(2):1104-1112.
[14] Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S. Shell cross-linked stearic acid grafted chitosan oligosacchande self-aggregated micelles for controlled release of paclitaxel [J]. Colloid Surf B, 2006, 50(2): 97-103.
[15] Sajeesh S, Sharma CP. Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery[J]. J Biomed Mater Res B, 2006, 76B: 298-305.
[16] Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW. Acetyl Histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: endocytosis, exocytosis and drug release [J]. J Control Release, 2006, 115(1): 37-45.
[17] Braud C, Vert M. Degradation of poly((3-malic acid)-monitoring of oligomers formation by aqueous SEC and HPCE [J]. Polym Bull, 1992, 29(1-2): 177-183.
[18] Osanai S, Nakamura K. Effects of complexation between liposome and poly(malic acid) on aggregation and leakage behaviour [J]. Biomaterials, 2000, 21(9):867-876.
[19] Cammas-Marion S, Bear MM, Harada A, Guerin P, Kataoka K. New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester [J]. Macromol Chem Phys, 2000, 201(3): 355-364.
[20] Lee BS, Fujita M, Khazenzon NM, Wawrowsky KA, Wachsmann-Hogiu S, Farkas DL, Black KL, Ljubimova JY, Holler E. Polycefin, a new prototype of a multifunctional nonconjugate based on poly(beta-L-malic acid) for drug delivery [J].Bioconjugate Chem, 2006, 17(2): 317-326.
[21] Otsuka H, Nagasaki Y, Kataoka K. PEGylated nanoparticles for biological and pharmaceutical applications [J]. Adv Drug Deliv Rev, 2003, 55(3): 403-419.
[22]Hu Y,Jiang XQ,Ding Y,Zhang LY,Yang CZ,Zhang JF,Chen JN,Yang YH.Preparation and drug release behaviors of nimodipine-loaded poly(caprolactone)-poly(ethylene oxide)-polylactide amphiphilic copolymer nanoparticles[J].Biomaterials,2003,24(13):2395-2404.
[23]Nakase I,Gallis B,Takatani-Nakase T,Oh S,Lacoste E,Singh NP,Goodlett DR,Tanaka S,Futaki S,Lai H,Sasaki T.Transferrin receptor-dependent cytotoxicity of artemisinin-transferring conjugates on prostate cancer cells and induction of apoptosis [J].Cancer Lett,2009,274(2):290-298.
[24]Oh S,Kim BJ,Singh NP,Lai H,Sasaki T.Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide[J].Cancer Lett,2009,274(1):33-39.
[25]Emonard H,Bellon G,Diesbach P,Mettlen M,Hornebeck W,Courtoy PJ.Regulation of matrix metalloproteinase(MMP) activity by the low-density lipoprotein receptor-related protein(LRP).A new function for an “old friend”[J].Biochimie,2005,87(3-4):369-376.
[26]Zhao WH,Zhang QY,Kang XM,Jin S,Lou CJ.AIBI is required for the acquisition of epithelial growth factor receptor-mediated tamoxifen resistance in breast cancer cells[J].Biochem Bioph Res Co,2009,380(3):699-704.
[27]Gabor F,Bogner E,Weissenboeck A,Wirth M.The lectin-cell interaction and its implications to intestinal lectin-mediated drug delivery[J].Adv Drug Deliv Rev,2004,56(4):459-480:
[28]Leamon CP,Low PS.Delivery of macromolecules into living cells:a method that exploits folate receptor endocytosis[J].P Natl Acad Sci USA,1991,88(13):5572-5576.
[29]Pan J,Feng SS.Targeted delivery of paclitaxel using folate-decorated poly(lactide) evitamin E TPGS nanoparticles[J].Biomaterials,2008,29(17):2663-2672.
[30]Sakahara H,Saga T.Avidin-biotin system for delivery of diagnostic agents[J].Adv Drug Deliv Rev,1999,37(1-3):89-101.
[31]Correa-Gonzalez L,de Murphy CA,Ferro-Flores G,Pedraza-Lopez M,Murphy-Stack E,Mino-Leon D,Perez-Villasenor G,Diaz-Torres Y,Munoz-Olvera R.Uptake of ~(153)Sm-DAPA-bis-biotin and ~(99m)Tc-DTPA-bis-biotin in rat AS-30D-hepatoma cells[J].Nucl Med Biol,2003,30(2):135-140.
[32]Na K,Lee TB,Park KH,Shin EK,Lee YB,Choi HK.Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as at argeted anti-cancer drug delivery system [J]. Eur JPharm Sci, 2003, 18(2): 165-173.
[33] Negishi T, Koizumi F, Uchino F, Kuroda J, Kawaguchi T, Naito S, Matsumura Y. NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel [J]. Brit J Cancer, 2006, 95(5):601-606.
[34] Soepenberg O, Sparreboom A, Jonge MJA, Planting AST, Heus G, Loos WJ,Hartman CM, Bowden C, Verweij J. Real-time pharmacokinetics guiding clinical decisions: phase I study of a weekly schedule of liposome with solid encapsulated paclitaxel in patients tumours [J]. Eur J Cancer, 2004, 40(5): 681-688.
[35] You JO, Auguste DT. Feedback-regulated paclitaxel delivery based on poly(N,N-dimethylaminoethyl methacrylate-co-2-Hydroxyethyl methacrylate) nanoparticles [J]. Biomaterials, 2008, 29(12): 1950-1957.
[1]Service RF.Nanotechnology takes aim at cancer[J].Science,2005,310(5751):1132-1134.
[2]LaVan DA,McGuire T,Langer R.Small-scale systems for in vivo drug delivery [J].Nat Biotechnol,2003,21(10):1184-1191.
[3]LaVan DA,Lynn DM,Langer R.Moving smaller in drug discovery and delivery [J].Nat Rev Drug Discov,2002,1(1):77-84.
[4]Lesniak MS,Brem H.Targeted therapy for brain tumors[J].Nat Rev Drug Discov,2004,3(6):499-508.
[5]Chen ML.Lipid excipients and delivery systems for pharmaceutical development:A regulatory perspective[J].Adv Drug Deliv Rev,2008,60(10):768-777.
[6]蒋挺大.壳聚糖(第二版)[M].北京:化学工业出版社,2006:12.
[7]Harris JM,Struck EC,Case MG,Paley MS,Vanalstine JM,Brookes DE.Synthesis and characterization of poly(ethylene glycol) derivatives[J].J Polym Sci Pol Chem,1984,22(2):341-352.
[8]Kajiyama T,Kobayashi H,Taguchi T,Kataoka K,Tanaka J.Improved synthesis with high yield and increased molecular weight of poly(α,β-malic acid) by direct polycondensation[J].Biomacromolecules,2004,5(1):169-174.
[9]Portilla-Arias JA,Garcia-Alvarez M,de Ilarduya AM,Holler E,Munoz-Guerra S.Thermal decomposition of fungal poly(β,L-malic acid) and poly(β,L-malate)s[J].Biomacromolecules,2006,7(11):3283-3290.
[10]Vert M.Chemical routes to poly(β-malic acid) and potential applications of this water-soluble bioresorbable poly(β-hydroxy alkanoate)[J].Polym Degrad Stabil,1998,59(1-3):169-175.
[11]Coulembier O,Degee P,Hedrick JL,Dubois P.From controlled ring-opening polymerization to biodegradable aliphatic polyester Especially poly(β-malic acid)derivatives[J].Prog Polym Sci,2006,31(8):723-747.
[12]Guering P,Vert M,Braud C,Lenz RW.Optically active poly(β-malic acid)[J].Polym Bull,1985,14(2):187-192.
[13]Fukuzaki H,Yoshida M,Asano M,Kumakura M.Synthesis of copoly (D,L-lactic acid) with relatively low-molecular weight and in vitro degradation[J].Eur Polym J,1989,25(10):1019-1026.
[14]Imasaka K,Nagai T,Yoshida M,Fukuzaki H,Asano M,Kumakura M.Direct copolycondensation of epsilon-caprolactone with delta-valerolactone in the absence of catalysts.Eur Polym J,1990,26(8):831-836.
[15]解德良,姜标,杨昌正.羟基酸引发ε-己内酯开环聚合的研究[J].高分子学报,2000,5:532-537.
[16]李玉宝,生物医学材料[M].北京:化学工业出版社,2003:211.
[17]Sashiwa H,Kawasaki N,Nakayama A,Muraki E,Yamamoto N,Zhu H,Nagano H,Omura Y,Saimoto H,Shigemasa Y,Aiba SI.Chemical modification of chitosan 13Synthesis of organosoluble,palladium adsorbable,and biodegradable chitosan derivatives toward the chemical plating on plastics[J].Biomacromolecules,2002,3(5):1120-1125.
[18]Llanos GR,Sefton MV.Immobilization of poly(ethylene glycol) onto a poly(vinyl alcohol) hydrogel[J].Macromolecules,1991,24(23):6065-6072.
[19]Samuels RJ.Solid state characterization of the structure of chitosan films[J].J Polym Sci Pol Phys,1981,19(7):1081-1105.
[20]Ganji F,Abdekhodaie MJ.Synthesis and characterization of a new thermosensitive chitosan-PEG diblock copolymer[J].Carbohyd Polym,2008,74(3):435-441.
[21]Gupta KC,Jabrail FH.Effects of degree of deacetylation and cross-linking on physical characteristics,swelling and release behavior of chitosan microspheres[J].Carbohyd Polym,2006,66(1):43-54.
[1]Schmid EF,Smith DA.R&D technology investments:misguided and expensive or a better way to discover medicines?[J].Drug Discov Today,2006,11(17-18):775-784.
[2]Allen TM,Cullis PR.Drug delivery system:Entering the mainstream[J].Science,2004,303(5665):1818-1822.
[3]Duncan R.The dawning era of polymer therapeutics[J].Nat Rev Drug Discov,2003,2(5):347-360.
[4]Shiladitya S,David E,Ishan C,Zhao G,Nichi W,Tanyel K,Ram S.Temporal targeting of tumor cells and neovasculature with a nanoscale delivery system[J].Nature,2005,436(7050):568-572.
[5]Miwa A,Ishibe A,Nakano M,Yamahira T,Itai S,Jinno S,Kawahara H.Development of novel chitosan derivatives as micellar carriers of Taxol[J].Pharm Res,1998,15(12):1844-1850.
[6]Liu CG,Desai KGH,Chen XG,Park HJ.Linoleic acid-modified chitosan for formation of self-assembled nanoparticles[J].J Agr Food Chem,2005,53(2):437-441.
[7]Park JH,Kwon SG,Nam JO,Park RW,Chung H,Seo SB,Kim IS,Kwon IC,Jeong SY.Self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid for RGD peptide delivery[J].J Control Release,2004,95(3):579-588.
[8]Zhang C,Ping QN,Zhang HJ,Shen J.Preparation of N-alkyl-O-sulfate chitosan derivatives and micellar solubilization of taxol[J].Carbohyd Polym,2003,54(2):137-141.
[9]Yang XD,Zhang QQ,Wang YS,Chen H,Zhang HZ,Gao FP,Liu LR.Self-aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: Synthesis; characterization; aggregation and methotrexate release in vitro [J]. Colloid Surf B,2008,61(2): 125-131.
[10] Liu ZH, Jiao YP, Wang YF, Zhou CR, Zhang ZY. Polysaccharides-based nanoparticles as drug delivery systems [J]. Adv Drug Deliv Rev, 2008, 60(15): 1650-1662.
[11] Prabahran M, Grailer JJ, Pilla S, Steeber DA, Gong SQ. Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn~® H40, poly(1-lactide) and poly (ethylene glycol) for tumor-targeted drug delivery [J]. Biomaterials, 2009, 30 (16): 3009-3019.
[12] Seo JH, Moon HS, Kim IY, Guo DD, Lee HG, Choi YJ, Cho CS. PEGylated conjugated linoleic acid stimulation of apoptosis via a p53-mediated signaling pathway in MCF-7 breastcancer cells [J]. Eur J Pharm Biopharm, 2008, 70 (2): 621-626.
[13] Pyrz WD, Buttrey DJ. Particle size determination using TEM: A discussion of image acquisition and analysis for the novice microscopist [J]. Langmuir, 2008, 24(20):11350-11360.
[14] Park JS, Han TH, Lee KY, Han SS, Hwang JJ, Moon DH, Kim SY, Cho YW. N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs: Endocytosis, exocytosis and drug release [J]. J Control Release, 2006, 115 (1): 37-45.
[15] Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, Kim Y, Park JH, Kim J, Her S, Oh YK, Kwon IC, Kim K, Jeon SY. Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles [J]. J Control Release, 2009, 13 (3): 259-267.
[16] Li FQ, Su H, Wang J, Liu JY, Zhu QG, Fei YB, Pan YH, Hu JH. Preparation and characterization of sodium ferulate entrapped bovine serum albumin nanoparticles for liver targeting [J]. Int J Pharm, 2008, 349 (1-2): 274-282.
[17] Li SD, Huang L. Pharmacokinetics and biodistribution of nanoparticles [J]. Mol Pharm, 2008, 5 (4): 496-504.
[18] Dobrovolskaia MA, Aggarwal P, Hall JB, Mcneil SE. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution [J]. Mol Pharm, 2008, 5 (4): 487-495.
[19] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles [J]. Mol Pharm, 2008, 5 (4): 505-515.
[20] Manjunath K, Venkateswarlu. Pharmacokinetics, tissue distribution and bioavailability of clozapine solid lipid nanoparticles after intravenous and intraduodenal administration [J]. J Control Release, 2005, 107 (2): 215-228.
[21] Dutta P, Dey J, Ghosh G, Nayak RR. Self-association and microenvironment of random
amphiphilic copolymers of sodium N-acryloyl-L-valinate and N-dodecylacrylamide in
aqueous solution [J]. Polymer, 2009, 50 (6): 1516-1525.
[22] Deepa S, Mishra AK. Fluorescence spectroscopic study of serum albumin-bromadiolone
interaction: fluorimetric determination of bromadiolone [J]. J Pharmaceut Biomed, 2005, 38
(3): 556-563.
[23] Fuguet E, Rafols C, Roses M, Bosch E. Critical micelle concentration of surfactants in
aqueous buffered and unbuffered systems [J]. Anal Chim Acta, 2005, 548(1-2): 95-100.
[24] Srivastava A, Waite JH, Stucky GD, Mikhailovsky A. Fluorescence investigations into
complex coacervation between polyvinylimidazole and sodium alginate [J]. Macromolecules,
2009, 42 (6): 2168-2176.
[25] Tan YL, Liu CG. Self-aggregated nanoparticles from linoleic acid modified
carboxymethyl chitosan: Synthesis, characterization and application in vitro [J]. Colloid Surf
B, 2009, 69 (2): 178-182.
[26] Danhier F, Lecouturier N, Vroman B, Jerome C, Marchand-brynaert J, Feron O, Preat V.
Paclitaxel-loaded PEGylated PLGA-based nanoparticles: In vitro and in vivo evaluation [J]. J
Control Release, 2009, 133 (1): 11-17.
[27] Jung J, Lee IH, Lee E, Park J, Jon S. pH-Sensitive polymer nanospheres for use as a
potential drug delivery vehicle [J]. Biomacromolecules, 2007, 8 (11): 3401-3407.
[28] Xu ZH, Chen LL, Gu WW, Gao Y, Lin LP, Zhang ZW, Zi Y, Li YP. The performance of
decetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma [J].
Biomaterials, 2009, 30 (2): 226-232.
[29] Hu FQ, Ren GF, Yuan H, Du YZ, Zeng S. Shell cross-linked stearic acid grafted chitosan
oligosaccharide self-aggregated micelles for controlled release of paclitaxel [J]. Colloid Surf
B, 2006, 50 (2): 97-103.
[30] Zhang ZP, Feng SS. The drug encapsulation efficiency, in vitro drug release, cellular
uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol
succinate nanoparticles [J]. Biomaterials, 2006, 27 (21): 4025-4033.
[31] Prokop A, Davidson JM. Nanovehicular intracellular delivery systems [J]. J Pharm Sci,
2008, 97(9): 3518-3590.
[32] Mohamed F, Van der Walle CF. Engineering biodegradable polyester particles with
specific drug targeting and drug release properties [J]. J Pharm Sci, 2008, 97(1): 71-87.
[33] Lee SJ, Park K, Oh YK, Kwon SH, Her S, Kim IS, Choi K, Lee SJ, Kim H, Lee SG,
Kim K, Kwon IC. Tumor specificity and therapeutic efficacy of photosensitizer-encapsulated
43 glycol chitosan-based nanoparticles in tumor-bearing mice[J].Biomaterials,2009,30(15):2929-2939.
[1]Davis HE,Chen Z,Shin DM.Nanoparticle therapeutics:an emerging treatment modality for cancer[J].Nat Rev Drug Discov,2008,7(9):771-782.
[2]王晓华.生物化学与分子生物学实验技术[M].北京:化学工业出版社,2008:74-75.
[3]张忠泉,陈百泉.药理学实验指导[M].郑州:郑州大学出版社,2007:21.
[4]张馨欣,甘勇,杨星钢,朱春柳,甘莉,聂淑芳,潘卫三.聚乙二醇修饰的羟基喜树碱纳米粒脂质载体的制备及小鼠组织分布[J].药学学报,2008,43(1):91-96
[5]Williams J,Lansdown R,Sweitzer R,Romanowski M,LaBell R,Ramaswami R,Unger E.Nanoparticle drug delivery system for intravenous delivery of topoisomerase inhibitors[J].J Control Release,2003,91(1-2):167-172.
[6]Liang XJ,Shen DW,Garfield S,Gottesman MM.Mislocalization of membrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines[J].Cancer Res,2003,63(18):5909-5916.
[7]Zhang LY,Yang M,Wang Q,Li Y,Guo R,Jiang XQ,Yang CZ,Liu BR.10-Hydroxycamptothecin loaded nanoparticles:Preparation and antitumor activity in mice[J].J Control Release,2007,112(2):153-162.
[8]Mao S,Germershaus O,Fischer D,Linn T,Schnepf R,Kissel T.Uptake and transport of PEG-graft-trimethyl-chitosan copolymer-insulin nanocomplexes by epithelial cells[J].Pharm Res,2005,22(12):2058-2068.
[9]Jae HP,Seulki L,Jong-Ho K,Kyeongsoon P,Kwangmeyung K,Ick CK.Polymeric nanomedicine for cancer therapy[J].Prog Polym Sci,2008,33(1): 113-137.
[10]Nam HY,Kwon SM,Chung H,Lee SY,Kwon SH,Jeon H,Kim Y,Park JH,Kim J,Her S,Oh YK,Kwon IC,Kim K,Jeong SY.Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles[J].J Control Release,2009,135(3):259-267.
[11]Yakashi Y,Yoshiyuki H,Motoki H,Kimiko K,Yoshie M.Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts[J].Eur J Pharm Biopharm,2008,70(3):718-725.
[12]Cristina L Z,William TP,Anuradha S.Use of avidin/biotin-liposome system for enhanced peritoneal drug delivery in an ovarian cancer model[J].Int J Pharm,2007,337(1/2):316-328.
[13]Hilgenbrink AR,Low PS.Folate receptor-mediated drug targeting from therapeutics to diagnostics[J].J Pharm Sci,2005,94(10):2135-2146.
[14]Patil YB,Toti US,Khdair A,Mac L,Panyamc J.Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery[J].Biomaterials,2009,30(5):859-866.
[15]张丽珺,方晓玲.聚合物纳米粒和胶束的主动靶向研究进展[J].国外医学药学分册,2005,32(6):399-403.
[16]Kimura M,Umegaki K,Higuchi M,Thomas P,Fenech M.Methylenetetrahydrofolate reductase C677T polymorphism,folic acid and riboflavin are important determinants of genome stability in cultured human lymphocytes[J].J Nutr,2004,134(1):48-56.
[17]Mock DM,Lankford GL,Mock NI.Biotin accounts for only half of the total avidin-binding substances in human serum[J].J Nutr,1995,125(4):941-946.
[18]Gregory RJ,Kirsten MT,John ME,John R,David N.Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumors[J].J Inorg Biochem,2004,98(10):1625-1633.
[19]中国药典 2005 版二部附录 XIXB.
[20]Yamamoto Y,Nagasaki Y,Kato Y,Sugiyama Y,Kataoka K.Long-circulating poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles with modulated surface charge[J].J Control Release,2001,77(1-2):27-38.
[21]Romberg B,Hennink WE,Storm G Sheddable coatings for long-circulating nanoparticles[J].Pharm Res,2008,25(1):55-71.
[22]Abraham AM,Walubo A.The effect of surface charge on the disposition of liposome-encapsulated gentamicin to the rat liver,brain,lungs and kidneys after intraperitoneal administration[J].Int J Antimicrob Ag,2005,25(5):392-397.
[23]Yamamoto H,Kuno Y,Sugimoto S,Takeuchi H,Kawashima Y.Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions[J].J Control Release,2005,102(2):373-381.
[24]Russell-Jones G,Mc Tavish K,McEwan J,Rice J,Nowotnik D.Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours[J].J Inorg Biochem,2004,98(10):1625-1633.
[25]Maeda H,Wu J,Sawa T,Matsumura Y.Hori K.Tumor vascular permeability and the EPR effect in macromolecular therapeutics:a review[J].J Control Release,2000,65(1-2):271-284.
[26]方超,裴元英.运载抗肿瘤药物的隐形纳米粒的研究进展[J].中国药学杂志,2004,39(2):89-92.
[27]Corrocher R,Pachor ML,Bambara LM,De Sandre G.Evidence for a folic acid binding protein in human cell membrane[J].Acta Haematol,1981,66(3):202-209.
[28]McGuigan AP,Sefton MV.The influence of biomaterials on endothelial cell thrombogenicity[J].Biomaterials,2007,28(16):2547-2571.
[29]GB/T 16886.122001.医疗器械生物学评价第1部分:评价与试验[S].北京:中国标准出版社,2002.
[30]Mayer A,Vadon M,Rinner B,Novak A,Wintersteiger R,Fr(o|¨)hlich E.The role of nanoparticle size in hemocompatibility[J].Toxicol,2009,258(2-3):139-147.
[1]Hans ML,Lowman AM.Biodegradable nanoparticles for drug delivery and targeting[J].Curr Opin Solid St M,2002,6(4):319-327.
[2]Liu WG,Sun SJ,Zhang X,De YK.Self-aggregation behavior of alkylated chitosan and its effect on the release of a hydrophobic drug[J].J Biomater Sci Polymer Edn,2003,14(8):851-859.
[3]Liu CG,Desai KGH,Chen XG,Park HJ.Linolenic acid-modified chitosan for formation of self-assembled nanoparticles[J].J Agric Food Chem,2005,53(2):437-441.
[4]Zhang J,Chen XG,Li YY,Liu CS.Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin[J].Nanomedicine,2007,3(4):258-265.
[5] Jiang GB, Quan DP , Liao KR, Wang HH. Preparation of polymeric micelles
based on chitosan bearing a small amount of highly hydrophobic groups [J].
Carbohyd Polym, 2006, 66(4): 514-520.
[6] Lee KY, Jo WH, Kwon IC, Kim YH, Jeong SY. Structural determination and
interior polarity of self-aggregates prepared from deoxycholic acid-modified chitosan
in water [J]. Macromolecules, 1998, 31(2): 378-383.
[7] Park JK, Kim DG, Choi C, Jeong YL, Kim MY, Jang MK , Nah JW. Preparation
and characterization of lithocholic acid conjugated chitosan oligosaccharide
nanoparticles for hydrophobic anticancer agent carriers [J]. Polym-Korea, 2008, 32(3):
263-269.
[8] Wang YS, Liu LR, Jiang Q, Zhang QQ. Self-aggregated nanoparticles of
cholesterol-modified chitosan conjugate as a novel carrier of epirubicin [J]. Eur
Polym J, 2007, 43(1): 43-51.
[9] Natalija C, Ricardas M. Synthesis and study of water-soluble
chitosan-O-poly(ethylene glycol) graft copolymers [J]. Eur Polym J, 2004, 40(4):
685-691.
[10] Park JS, Koh YS, Bang JY , Jeong YI, Lee JJ. Antitumor effect of all-trans
retinoic acid-encapsulated nanoparticles of methoxy poly(ethylene glycol)-conjugated
chitosan against CT-26 colon carcinoma in vitro [J]. J Pharm Sci-US, 2008, 97(9):
4011-4019.
[11] Yan CY, Chen DW, Gu JW, Qin J. Nanoparticles of 5-fluorouracil (5-FU) loaded
N-succinyl-chitosan (Suc-Chi) for cancer chemotherapy: preparation, characterization
in vitro drug release and anti-tumour activity [J]. J Pharm Pharmacol, 2006, 58(9):
1177-1181.
[12] Kim K, Kwon S, Park JH, Chung H, Jeong SY, Kwon IC, Kim IS.
Physicochemical characterizations of self-assembled nanoparticles of glycol
chitosan-deoxycholic acid conjugates [J]. Biomacromolecules, 2005, 6(2): 1154-1158.
[13] Park JH, Kwon S, Nam JO, Park RW, Chung H, Seo SB, Kim IS, Kwon IC,
Jeong SY. Self-assembled nanoparticles based on glycol chitosan bearing 5(3-cholanic
acid for RGD peptide delivery [J]. J Control Release, 2004, 95(3): 579-588.
[14] Hwang HY, Kim IS, Kwon IC , Kim YH . Tumor targetability and antitumor
effect of docetaxel-loaded hydrophobically modified glycol chitosan nanoparticles [J].
J Control Release, 2008, 128(1): 23-31.
[15] Zhang C, Ping QN, Zhang HJ, Shen J. Preparation of N-alkyl-O-sulfate chitosan
91 derivatives and micellar solubilization of taxol[J].Carbohyd Polym,2003,54(4):137-141.
[16]Qian F,Cui FY,Ding JY,Tang C,Yin CH.Chitosan graft copolymer nanoparticles for oral protein drug delivery:preparation and characterization[J].Biomacromolecules,2006,7(10):2722-2727.
[17]Sajeesh S,Sharma CP.Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery[J].J Biomed Mater Res B,2006,76B(2):298-305.
[18]Park JS,Han TH,Lee KY,Han SS,Hwang JJ,Moon DH,Kim SY,Cho YW.N-acetyl histidine-conjugated glycol chitosan self-assembled nanoparticles for intracytoplasmic delivery of drugs:endocytosis,exocytosis and drug release[J].J Control Release,2006,115(1):37-45.
[19]Liu ZH,Jiao YP,Liu F,Zhang ZY.Heparin/chitosan nanoparticle carriers prepared by polyelectrolyte complexation[J].J Biomed Mater Res A,2007,83A(3):806-812.
[20]Chen Y,Mohanraj VJ,Parkin JE.Chitosan-dextran sulfate nanoparticles for delivery of an anti-angiogenesis peptide[J].Lett Pept Sci,2003,10(5-6):621-629.
[21]Bodnar M,Hartmann JF,Borbely J.Preparation and characterization of chitosan-based nanoparticles[J].Biomacromolecules,2005,6(5):2521-2527.
[22]Janes KA,Calvo P,Alonso MJ.Polysaccharide colloidal particles as delivery systems for macromolecules[J].Adv Drug Deliv Rev,2001,47(1):83-97.
[23]Gan Q,Wang T,Cochrane C,McCarron P.Modulation of surface charge,particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery[J].Colloid Surf B,2005,44(2-3):65-73.
[24]Guan XP,Quan DP,Liao KR,Wang T,Xiang P,Mai KC.Preparation and characterization of cationic chitosan-modified poly(D,L-lactide-co-glycolide)copolymer nanospheres as DNA carriers[J].J Biomater Appl,2008,22(4):353-371.
[25]李培,朱爱梅,刘庆林.沉淀聚合法制备壳聚糖磁性微球[J].化工时刊,2008,22(7):1-4.
[26]Yu JM,Li YH,Qiu LY,Jin Y.Self-aggregated nanoparticles of cholesterol-modified glycol chitosan conjugate:Preparation,characterization,and preliminary assessment as a new drug delivery carrier[J].Eur Polym J,2008,44(3):555-565.
[27]Yoo HS,Lee JE,Chung H,Kwon IC,Jeong SY.Self-assembled nanoparticles containing hydrophobically modified glycol chitosan for gene delivery[J].J Control Release,2005,103(1):235-243.
[28]Qi LF,Xu ZR.In vivo antitumor activity of chitosan nanoparticles[J].Bioorg Med Chem Lett,2006,16(16):4243-4245.
[29]Bilensoy E,Sarisozen C,Esendagli G,Dogan AL,Aktas Y,Sen M,Mungan NA.Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors[J].Int J Pharm,2009,371(1-2):170-176.
[30]Lee SJ,Park K,Oh YK,Kwon SH,Her S,Kim IS,Choi K,Lee SJ,Kim H,Lee SG,Kim K,Kwon IC.Tumor specificity and therapeutic efficacy of photosensitizer-encapsulated glycol chitosan-based nanoparticles in tumor-bearing mice[J].Biomaterials,2009,30(15):2929-2939.
[31]Min KH,Park K,Kim YS,Bae SM,Lee S,Jo HG,Park RW,Kim IS,Jeong SY,Kim K,Kwon IC.Hydrophobically modified glycol chitosan nanoparticles encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy[J].J Control Release,2008,127(3):208-218.
[32]Tan YL,Liu CG.Self-aggregated nanoparticles from linoleic acid modified carboxymethyl chitosan:Synthesis,characterizaton and applicatioin in vitro[J].Colloid Surf B,2009,69(2):178-182.
[33]Zhang J,Chen XG,Liu CS,Park HJ.Investigation of polymeric amphiphilic nanoparticles as antitumor drug carriers[J].J Mater Sci-Mater M,2009,20(4):991-999.
[34]Sarmento B,Ribeiro A,Veiga F,Ferreira D,Neufeld R.Oral bioavailability of insulin contained in polysaccharide nanoparticles[J].Biomacromolecules,2007,8(10):3054-3060.
[35]Chert F,Zhang ZR,Yuan F,Qin X,Wang MT,Huang Y.In vitro and in vivo study of N-trimethyl chitosan nanoparticles for oral protein delivery[J].Int J Pharm,2008,349(1-2):226-233.
[36]胡思隽,谢勇.壳聚糖纳米粒在基因治疗中的应用[J].国际病理科学与临床杂志,2006,26(2):150-152.
[37]Zhao Z,Yu SB,Wu FL,Mao ZB,Yu CL.Transfection of primary chondrocytes using chitosan-pEGFP nanoparticles[J].J Control Release,2006,112(2):223-228.
[38]Guliyeva U,Oner F,Ozsoy S,Haziroglu R.Chitosan microparticles containing plasmid DNA as potential oral gene delivery system[J].Eur J Pharm Biopharm,2006,62(1):17-25.
[39]Liu XD,Howard KA,Dong MD,Andersen MO,Rahbek UL,Johnsen MG,Hansen OC,Besenbacher F,Kjems J.The influence of polymeric properties on chitosan/siRNA nanoparticle formulation and gene silencing[J].Biomaterials,2007,28(6):1280-1288.
[40]王琳玲.壳聚糖及其衍生物抗菌性质的研究进展[J]_上海生物医学工程,2006,27(2):111-114.
[41]Motwani SK,Chopra S,Talegaonkar S,Kohli K,Ahmad FJ,Khar RK.Chitosan-sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery:Formulation,optimization and in vitro characterization[J].Eur J Pharm Biopharm,2008,68(3):513-525.
[42]Kim EM,Jeong HJ,Kim SL,Sohn MH,Nah JW,Born HS,Park IK,Cho CS.Asialoglycoprotein-receptor-targeted hepatocyte imaging using Tc-99m galactosylated chitosan[J].Nucl Med Biol,2006,33(4):529-534.
[43]Rhee K,Moon HJ,Park KS,Khang G.Evaluation of peptides tagged nanoparticle adhesion to activated endothelial cells[A].Jarm T,Kramar P,Zupanic A.11~(th) Mediteranean Conference On Medical And Biological Engineering And Computing 2007[C].Ljubljana,Slovenia,2007:835-838.
[44]Aktas Y,Yemisci M,Andrieux K,Gursoy RN,Alonso MJ,Fernandez-Megia E,Novoa-Carballal R,Quinoa E,Riguera R,Sargon MF,Celik HH,Demir AS,Hincal AA,Dalkara T,Capan Y,Couvreur P.Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26[J].Bioconjugate Chem,2005,16(6):1503-1511
[45]Jain A,Jain SK.In vitro and cell uptake studies for targeting of ligand anchored nanoparticles for colon tumors[J].Eur J Pharm Sci,2008,35(5):404-416.
[46]Fernandes JC,Wang HJ,Jreyssaty C,Benderdour M,Lavigne P,Qiu XP,Winnik FM,Zhang XL,Dai KR,Shi Q.Bone-protective effects of nonviral gene therapy with folate-chitosan DNA nanoparticle containing interleukin-1 receptor antagonist gene in rats with adjuvant-induced arthritis[J].Mol Ther,2008,16(7):1243-1251.
[47]姚倩,侯世祥,张瑄,赵钢,荀小军,游金宗.生物素化壳聚糖纳米粒的制备及其相关性质[J].药学学报,2007,42(5):557-561.