Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.     

Design of novel polysaccharidic nanostructures for gene delivery

The goal of the present work was to develop a new synthetic nanosystem for gene delivery. For this purpose, we chose two polysaccharides, hyaluronic acid (HA) and chitosan (CS), as the main components of the nanocarrier. Nanoparticles with different hyaluronate:chitosan (HA:CS) mass ratios (0.5:1 and 1:1) and different polymer molecular weights (hyaluronate 170 (HA) or <10?kDa (HAO) and chitosan 125 (CS) or 10-12?(CSO)?kDa) could be obtained using an ionic crosslinking method. These nanoparticles were loaded with pDNA and characterized for their size, zeta potential and pDNA association efficiency. Moreover, their toxicity and ability to transfect the model plasmid pEGFP-C1 were evaluated in the cell line HEK 293, as well as their intracellular fate. The results showed that HA:CS nanoparticles have a small size in the range of 110-230?nm, a positive zeta potential of +10 to +32?mV and a very high pDNA association efficiency of 87-99% (w/w). On the other hand, nanoparticles exhibited low cell toxicity and transfection levels up to 25% GFP expressing HEK?293 cells, lasting for the whole observation period of 10 days. We also provide basic information about the role of both polymers, HA and CS, and the effect of their molecular weight on the effectiveness of the resulting DNA nanocarrier, being the highest transfection levels observed with HAO:CSO 1:1 nanoparticles. In?conclusion, HA:CS nanoparticles are promising carriers for gene delivery.     

Facile synthesis of multi‐walled carbon nanotube via folic acid grafted nanoparticle for precise delivery of doxorubicin

The motive of work was to develop a multi‐walled carbon nanoplatform through facile method for transportation of potential anticancer drug doxorubicin (DOX). Folic acid (FA)‐ethylene diamine (EDA) anchored and acid functionalised MWCNTs were covalently grafted with DOX via π–π stacking interaction. The resultant composite was corroborated by ¹ H NMR, FTIR, XRD, EDX, SEM, and DSC study. The drug entrapment efficiency of FA‐conjugated MWCNT was found high and stability study revealed its suitability in biological system. FA‐EDA‐MWCNTs‐DOX conjugate demonstrated a significant in vitro anticancer activity on human breast cancer MCF‐7 cells. MTT study revealed the lesser cytotoxicity of folate‐conjugated MWCNTs. The obtained results demonstrated the targeting specificity of FA‐conjugate via overexpressed folate receptor deemed greater scientific value to overcome multidrug protection during cancer therapy. The proposed strategy is a gentle contribution towards development of biocompatible targeted drug delivery and offers potential to address the current challenges in cancer therapy.Inspec keywords: toxicology, nanoparticles, biomedical materials, scanning electron microscopy, drug delivery systems, nanofabrication, nanomedicine, nanocomposites, cellular biophysics, cancer, drugs, multi‐wall carbon nanotubes, Fourier transform infrared spectra, X‐ray chemical analysis, differential scanning calorimetry, proton magnetic resonance, organic compoundsOther keywords: facile synthesis, multiwalled carbon nanotube, precise delivery, multiwalled carbon nanoplatform, drug entrapment efficiency, FA‐conjugated MWCNT, stability study, biological system, human breast cancer MCF‐7 cells, MTT study, folate‐conjugated MWCNTs, overexpressed folate receptor, cancer therapy, biocompatible targeted drug delivery, anticancer drug doxorubicin, π‐π stacking interaction, composite material, ¹ H NMR, in vitro anticancer activity, folic acid grafted nanoparticle, folic acid‐ethylene diamine, acid functionalised MWCNT, FTIR spectra, XRD, EDX, SEM, FA‐EDA‐MWCNT‐DOX conjugate, cytotoxicity, DSC, C     

Evaluation of PEG-transferrin-PEI nanocomplex as a gene delivery agent

Polyethylenimine (PEI) has been shown to be an efficient nonviral delivery vector. To improve its specificity and reduce its cytotoxicity, PEI should be modified. Transferrin (Tf) is a cell-binding ligand and Tf-receptors are expressed in malignant cells. Modification of cationic polymer by polyethylene glycol (PEG) can reduce the protein interaction and cell cytotoxicity of delivery vectors. We have synthesized PEG-Tf-PEI conjugate as an efficient and safe carrier of plasmid DNA (pDNA). Nanocomplexes of conjugates with pDNA were characterized by measuring the particle size and the surface charge. Transfection efficiency of nanocomplexes in Jurkat cells was improved and cytotoxicity was decreased compared with those of PEI complex. This was due to a reduction in the membrane damaging effect via shielding of the positive charge on the nanocomplex surface by PEG.     

Quaternized chitosan/rectorite intercalative materials for a gene delivery system

Non-viral vectors have gained increasing attention in gene therapy because of their safety, but with the shortcoming of low transfection efficiency. We have developed a hybrid material as a novel non-viral vector, which combines the advantages of both biopolymer and clay in a gene delivery system. Quaternized chitosan was intercalated into the interlayers of rectorite to obtain a new polymer/layered silicate nanocomposite. In vitro and in vivo toxicity studies revealed that the nanocomposites were biocompatible and non-toxic. At the nanocomposite:pDNA mass ratio of 8:1, they achieved 100% pDNA adsorption capacity. In vitro cell transfection revealed a transfection efficiency of 32.1% at 96?h as shown by a flow-cytometric study, and the intensive green fluorescence protein (GFP) expression could last for up to 120?h. Furthermore, an in vivo transfection study showed that the most prominent GFP expression was observed in the gastric and duodenum mucosa, and good transfection efficiency was also obtained when injected into the muscle. All the results suggest that quaternized chitosan/rectorite nanocomposite is a novel and potential non-viral gene carrier.     

Targeted Theranostic Nano Vehicle Endorsed with Self‐Destruction and Immunostimulatory Features to Circumvent Drug Resistance and Wipe‐Out Tumor Reinitiating Cancer Stem Cells

The downsides of conventional cancer monotherapies are profound and enormously consequential, as drug‐resistant cancer cells and cancer stem cells (CSC) are typically not eliminated. Here, a targeted theranostic nano vehicle (TTNV) is designed using manganese‐doped mesoporous silica nanoparticle with an ideal surface area and pore volume for co‐loading an optimized ratio of antineoplastic doxorubicin and a drug efflux inhibitor tariquidar. This strategically framed TTNV is chemically conjugated with folic acid and hyaluronic acid as a dual‐targeting entity to promote folate receptor (FR) mediated cancer cells and CD44 mediated CSC uptake, respectively. Interestingly, surface‐enhanced Raman spectroscopy is exploited to evaluate the molecular changes associated with therapeutic progression. Tumor microenvironment selective biodegradation and immunostimulatory potential of the MSN‐Mn core are safeguarded with a chitosan coating which modulates the premature cargo release and accords biocompatibility. The superior antitumor response in FR‐positive syngeneic and CSC‐rich human xenograft murine models is associated with a tumor‐targeted biodistribution, favorable pharmacokinetics, and an appealing bioelimination pattern of the TTNV with no palpable signs of toxicity. This dual drug‐loaded nano vehicle offers a feasible approach for efficient cancer therapy by on demand cargo release in order to execute complete wipe‐out of tumor reinitiating cancer stem cells.     

Bio-functionalization of magnetite nanoparticles using an aminophosphonic acid coupling agent: new, ultradispersed, iron-oxide folate nanoconjugates for cancer-specific targeting

The present study describes a systematic approach towards the design and development of novel, bio-functionalized, magneto-fluorescent nanoparticles for cancer-specific targeting. Biocompatible, hydrophilic, magneto-fluorescent nanoparticles with surface-pendant amine, carboxyl or aldehyde groups, to be later used for bio-conjugation, were designed using an aminophosphonic acid coupling agent. These magneto-fluorescent nanoparticles were further functionalized with folic acid, using diverse conjugation strategies. A series of new iron-oxide folate nanoconjugates with excellent aqueous dispersion stability and reasonably good hydrodynamic sizes under a wide range of physiological conditions were developed. These ultradispersed nanosystems were analyzed for their physicochemical properties and cancer-cell targeting ability, facilitated by surface modification with folic acid. The nanoparticle size, charge, surface chemistry, magnetic properties and colloidal stability were extensively studied using a variety of complementary techniques. Confocal microscopy, performed with folate receptor positive human cervical HeLa cancer cells, established that these non-cytotoxic iron-oxide folate nanoconjugates were effectively internalized by the target cells through receptor-mediated endocytosis. Cell-uptake behaviors of nanoparticles, studied using magnetically activated cell sorting (MACS), clearly demonstrated that cells over-expressing the human folate receptor internalized a higher level of these nanoparticle-folate conjugates than negative control cells.     

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles

Dendritic cells (DCs) are professional antigen presenting cells that induce, sustain, and regulate immune responses. Gene modification of DCs is of particular interest for immunotherapy of diseases where the immunes system has failed or is abnormally regulated, such as in cancer or autoimmune disease. Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA. Among various non-viral vectors, chitosan is considered to be a good candidate for gene delivery system, however, lack of cell specificity and low transfection of chitosan need to be overcome prior to clinical use. In this study, mannosylated chitosan (MC) was prepared to induce the receptor-mediated endocytosis and targeting into antigen presenting cells (APCs), especially DCs having mannose receptors. MC showed great ability to form complexes with DNA and showed suitable physicochemical properties for gene delivery system. It had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell line than chitosan itself. Also, MC/DNA complex was more efficient for transferring IL-12 gene into DCs rather than water-soluble chitosan (WSC)/DNA one, which resulted in better induction of INF-gamma from DCs. Therefore, MC is a promising gene delivery system for repeated administration to maintain sustained gene expression, thereby opening the possibility for immunotherapy.     

In vitro evaluation of folic acid modified carboxymethyl chitosan nanoparticles loaded with doxorubicin for targeted delivery

The development of smart targeted nanoparticle that can deliver drugs to direct cancer cells, introduces better efficacy and lower toxicity for treatment. We report the development and characterizations of pH-sensitive carboxymethyl chitosan modified folic acid nanoparticles and manifest their feasibility as an effective targeted drug delivery vehicle. The nanoparticles have been synthesized from carboxymethyl chitosan with covalently bonded bifunctional 2,2′-(ethylenedioxy)-bis-(ethylamine) (EDBE) through the conjugation with folic acid. The conjugation has been analyzed by Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The resultant nanoparticles with an average size less then 200 nm measured by dynamic light scattering and transmission electron microscopy. Confocal microscopy and flow cytometric analysis have revealed that folate-mediated targeting significantly enhances the cellular uptake of the nanoparticle and thus facilitates apoptosis of cancer cells (HeLa, B16F1). For the application of the nanoparticles as a drug carrier, Doxorubicin a potent anticancer drug has been loaded into the nanoparticles, with the drug loading amount and the drug release pattern observed.     

Cytocompatible chitosan-graft-mPEG-based 5-fluorouracil-loaded polymeric nanoparticles for tumor-targeted drug delivery

Biodegradable materials like chitosan (CH) and methoxy polyethylene glycol (mPEG) are widely being used as drug delivery carriers for various therapeutic applications. In this study, copolymer (CH-g-mPEG) of CH and carboxylic acid terminated mPEG was synthesized by carbodiimide-mediated acid amine reaction. The resultant hydrophilic copolymer was characterized by Fourier transform infrared spectroscopy and ¹H NMR studies, revealing its relevant functional bands and proton peaks, respectively. Blank polymeric nanoparticles (B-PNPs) and 5-fluorouracil loaded polymeric nanoparticles (5-FU-PNPs) were formulated by ionic gelation method. Furthermore, folic acid functionalized FA-PNPs and FA-5-FU-PNPs were prepared for folate receptor-targeted drug delivery. FA-5-FU-PNPs were characterized by particle size, zeta potential, and in vitro drug release studies, resulting in 197.7?nm,?+29.9?mv, and sustained drug release of 88% in 24?h, respectively. Cytotoxicity studies were performed for FA-PNPs and FA-5-FU-PNPs in MCF-7 cell line, which exhibited a cell viability of 80 and 41%, respectively. In vitro internalization studies were carried out for 5-FU-PNPs and FA-5-FU-PNPs which demonstrated increased cellular uptake of FA-5-FU-PNPs by receptor-mediated transport. Significant (p?相似文献   

Synthesis, characterization and transfection of a novel folate-targeted multipolymeric nanoparticles for gene delivery

Novel folate-conjugated biodegradable multipolymeric nanoparticles (NPs) were constructed and evaluated for potential use in gene delivery to human cervical carcinomas Hela cells, which overexpressed folate receptors. Folate-poly(ethylene glycol)-poly(d, l-lactic-co-glycolic acid) (PELGA-F) was synthesized and collaborated with poly-l-lysine (PLL) to form polymer-polycationic peptide-DNA (PPD) NPs. Fluorescein sodium and polylysine-condensed DNA (PD) were encapsulated in both PELGA nanoparticles (PELGA-NPs) and folate modified nanoparticles (PELGA-F-NPs), which were prepared by a modified solvent extraction/evaporation method. Effects of the folate conjugation and PLL introduction on the uptake of NPs was qualified by fluorescent invert microscopy and quantified by spectrofluorometric measurement, while effect on the gene expression was measured by X-gal staining and luciferase assay, both using Hela cells as an in vitro model. Results showed that cellular uptake of NPs was enhanced by folate modification, but had no difference after PLL encapsulation. In transfection tests, increased gene expression also confirmed the different functions of folate and PLL introduction. It is feasible that folate-linked multipolymeric NPs should be an efficient targeted carrier for gene delivery.     

Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy applications

The encapsulation of DNA inside nanoparticles meant for gene delivery applications is a challenging process where several parameters need to be modulated in order to design nanocapsules with specific tailored characteristics. The purpose of this study was to investigate and improve the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using tripolyphosphate (TPP) as polyanionic crosslinker. Nanocapsule morphology and encapsulation efficiency were analyzed as a function of chitosan degree of deacetylation and chitosan-TPP ratio. The manipulation of these parameters influenced not only the particle size but also the encapsulation and release of pDNA. Consequently the transfection efficiency of the nanoparticulated systems was also enhanced with the optimization of the particle characteristics. Overall, the differently formulated nanoparticulated systems possess singular properties that can be employed according to the desired gene delivery application.     

An E3‐14.7K Peptide that Promotes Microtubules‐Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non‐viral vectors. Here, the interaction between E3‐14.7K and FIP‐1 to favor migration of pDNA along microtubules is exploited. E3‐14.7K is an early protein of human adenoviruses that interacts via FIP‐1 (Fourteen.7K Interacting Protein 1) protein with the light‐chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79‐98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non‐directional movements in the cytoplasm. Remarkably, P79‐98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.     

Synthesis and properties of a novel methoxy poly(ethylene glycol)-modified galactosylated chitosan derivative

Chitosan and its derivatives are attractive non-viral vectors. To produce target-cell specificity and improve the solubility of chitosan, a novel chitosan derivative, modified with galactose and methoxy poly(ethylene glycol) (mPEG) was synthesized, and structure changes of chitosan and its derivatives were characterized. Compared to chitosan, the solution viscosity of the novel chitosan derivative drastically decreased. And, the degree of substitution (DS) of chitosan by galactose and mPEG were calculated as 0.09 and 0.30. The average diameter and zeta potential of mPEGylated galactosylated chitosan (GaC) nanoparticle containing VRMFat plasmid were 178 nm and +2.93 mV, suggesting suitable properties for gene delivery system. The gel electrophoresis confirmed that the plasmid DNA was remained completely by the mPEGylated GaC nanoparticle. And, the cytotoxic effect of mPEGylated GaC nanoparticles on human embryonic kidney (HEK 293) cells was negligible in comparison with that of control chitosans. Therefore, it is expected that the mPEGylated GaC will have the potential as a targeting gene delivery system for a further application. Tao Zhang and Dong Li equally contributed to this research.     

Emergence of Gold‐Mesoporous Silica Hybrid Nanotheranostics: Dox‐Encoded,Folate Targeted Chemotherapy with Modulation of SERS Fingerprinting for Apoptosis Toward Tumor Eradication

Strategically fabricated theranostic nanocarrier delivery system is an unmet need in personalized medicine. Herein, this study reports a versatile folate receptor (FR) targeted nanoenvelope delivery system (TNEDS) fabricated with gold core silica shell followed by chitosan–folic acid conjugate surface functionalization by for precise loading of doxorubicin (Dox), resembled as Au@SiO₂‐Dox‐CS‐FA. TNEDS possesses up to 90% Dox loading efficiency and internalized through endocytosis pathway leading to pH and redox‐sensitive release kinetics. The superior FR‐targeted cytotoxicity is evaluated by the nanocarrier in comparison with US Food and Drug Administration (FDA)‐approved liposomal Dox conjugate, Lipodox. Moreover, TNEDS exhibits theranostic features through caspase‐mediated apoptosis and envisages high surface plasmon resonance enabling the nanoconstruct as a promising surface enhanced Raman scattering (SERS) nanotag. Minuscule changes in the biochemical components inside cells exerted by the TNEDS along with the Dox release are evaluated explicitly in a time‐dependent fashion using bimodal SERS/fluorescence nanoprobe. Finally, TNEDS displays superior antitumor response in FR‐positive ascites as well as solid tumor syngraft mouse models. Therefore, this futuristic TNEDS is expected to be a potential alternative as a clinically relevant theranostic nanomedicine to effectively combat neoplasia.     

Transfection efficiency of depolymerized chitosan and epidermal growth factor conjugated to chitosan–DNA polyplexes

An efficient non-viral gene delivery for varieties of cells has been considered essential for gene therapy and tissue engineering. This study evaluated transfection efficiency of chitosan (HW) with molecular weights (Mw) at 470 and degree of deacetylation (DDA) 80% and its depolymerization product (LW) with Mw at 16 kDa and DDA 54%, as well as epidermal growth factor (EGF) conjugated to chitosan–DNA microparticles of both HW and LW by using either disulfide linkage or NHS-PEO₄-Maleimide as a cross linker. The results revealed that the depolymerized LW at chitosan/DNA charge ratio 56:1 and pH 6.9 gave high transfection efficiency in both KB, a cancer cell line, and fibroblast cells at about the same level of Lipofectamine^TM, but the EGF-conjugated chitosan–DNA polyplexes from these methods did not improve transfection efficiency, which may come from the aggregation and fusing of the complexes as shown in scanning electron microscopy. However, this depolymerized LW chitosan showed the potential for further development as a safe and cost-effective non-viral gene delivery vehicle.     

Folate-conjugated zein/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanocomplexes for the enhancement of cellular uptake and cytotoxicity of gefitinib

Integrating various functional components into a single nano-platform is an ideal but challenging strategy for cancer therapy. Herein, a facile approach to fabricating multi-targeted nano-drug delivery systems which can be monitored is reported. Superparamagnetic iron oxide nanoparticles (SPIONs) and gefitinib (GEF) were encapsulated into folic acid-conjugated zein (Fa–zein) nanocomplexes (GEF-FSZs) with good dispersity, high GEF loading efficiency and pH-dependent release profile. The uptake of water-insoluble GEF was facilitated by encapsulating GEF into a zein-based nanocomplex, the magnetic responsive property endowed by SPIONs and the conjugation of Fa, which resulted in enhanced toxicity to A549 cells. The endocytosis study indicated that macropinocytosis and clathrin/caveolae-independent endocytosis exerted great influence on the internalization of GEF-FSZs. These results implied that GEF-FSZs could be a promising candidate for controlled and targeted drug delivery.     

Small-sized carbon nanohorns enabling cellular uptake control

Carbon nanotubes perform well in preclinical tests for drug delivery and diagnostic imaging, but controlling the size at less than 100 nm to avoid nonspecific uptake by reticuloendothelial systems while targeting delivery to cells of interest via receptor-mediated endocytosis is difficult, which currently limits their widespread use. Herein, 20-50-nm graphene tubules, small-sized single-walled carbon nanohorns (S-SWNHs), are obtained with a yield of 20% or higher by an oxidative exfoliation of 100 nm pristine SWNH aggregates. S-SWNHs are highly hydrophilic and remarkably resistant to cellular uptake by macrophages (RAW 264.7 cells), tumor cells (HeLa or KB), or normal cells (FHs 173We). The nonstimulatory property to cell membranes therefore makes cellular uptake control of S-SWNHs by functionalization easy. By attaching phospholipid polyethylene glycol, the cellular internalization of S-SWNHs is almost completely inhibited in RAW 264.7 macrophages. When functionalized with tumor-targeting folic acid (FA), FA-S-SWNHs are taken up by FA receptor-overexpressing KB cells but not by normal human embryonic cells (FHs 173We), which do not express the FA receptor. With a high rate of stealth and targeting in vitro, S-SWNHs are one of the most promising nanoparticles for medical use.     

叶酸协同EDC改性高脱乙酰度壳聚糖微球的制备及工艺探究

使用高脱乙酰度壳聚糖制备了靶向药物载体-叶酸改性壳聚糖微球。首先通过碱法制备了高脱乙酰度的壳聚糖,采用酸碱滴定法和傅里叶变换红外光谱(FTIR)对其结构进行了表征,结果表明,经脱乙酰化处理后的壳聚糖脱乙酰度高达93.8%。然后以高脱乙酰度壳聚糖制备了叶酸改性壳聚糖,创新性地发现用1∶1的二甲基亚砜和水的混合溶剂可以得到壳聚糖和叶酸反应的均相体系。通过不同的改性方案,得到了不同改性程度的壳聚糖,改性程度分别达到了2.60%、5.10%、8.75%和9.49%。最后用三聚磷酸钠交联制备了叶酸改性壳聚糖微球,并用激光粒度仪系统地分析了三聚磷酸钠和改性壳聚糖的用量比例及浓度对微球的粒径、Zeta电位的影响。研究发现,随着三聚磷酸钠与改性壳聚糖的比值增大,微球的粒径和Zeta电位都增大,当比值增加到一定的程度,微球的粒径会快速增加。对三聚磷酸钠和改性壳聚糖加入浓度的研究,发现浓度的增大将导致粒径的增大和Zeta电位的降低。