Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.     

Functionalized nanostructures with application in regenerative medicine

In the last decade, both regenerative medicine and nanotechnology have been broadly developed leading important advances in biomedical research as well as in clinical practice. The manipulation on the molecular level and the use of several functionalized nanoscaled materials has application in various fields of regenerative medicine including tissue engineering, cell therapy, diagnosis and drug and gene delivery. The themes covered in this review include nanoparticle systems for tracking transplanted stem cells, self-assembling peptides, nanoparticles for gene delivery into stem cells and biomimetic scaffolds useful for 2D and 3D tissue cell cultures, transplantation and clinical application.     

A combinatorial polymer library approach yields insight into nonviral gene delivery

The potential of gene therapy to benefit human health is tremendous because almost all human diseases have a genetic component, from untreatable monogenic disorders to cancer and heart disease. Unfortunately, a method for gene therapy that is both effective and safe has remained elusive. It has been said that "there are only three problems in gene therapy - delivery, delivery, and delivery." (quote from I. M. Verma in Jaroff, L. TIME, 1999; Jan 11). This Account describes an alternative strategy to viral gene delivery: the design of biodegradable polymers that are able to deliver DNA like a synthetic virus. Using high-throughput synthesis and screening techniques, we have created libraries of over 2000 structurally unique poly(β-amino esters) (PBAEs). PBAEs are formed by the conjugate addition of amines to diacrylates. These biomaterials are promising for nonviral gene delivery due to their ability to condense plasmid DNA into small and stable nanoparticles and their ability to promote cellular uptake and endosomal escape. Our laboratory has iteratively improved PBAE nanoparticles through polymer end modifications and nanoparticle coatings. Lead PBAEs have high gene delivery efficacy and low cytotoxicity both in vitro and in vivo. Certain polymer structural characteristics are important for effective gene delivery. The best PBAEs are linear polymers of ~10 kDa that contain hydroxyl side chains and primary amine end groups. These polymers bind DNA to form nanoparticles that are small (<200 nm) and stable and have near-neutral ζ potential in the presence of serum-containing media. Lead PBAEs also contain tertiary amines that can buffer the low pH environment of endosomes and facilitate escape of polymer/DNA particles into the cytoplasm. Diamine end-modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers (C32) bind DNA more tightly and form smaller nanoparticles than other PBAEs. These nanoparticles also have higher cellular uptake and the best gene expression of all gene delivery polymers in the library. These polymers are more effective for gene delivery than top commercially available nonviral vectors including jet-PEI and Lipofectamine 2000 and are comparable to adenovirus for in vitro gene delivery to human primary cells. In vivo, these PBAE/DNA particles are promising as cancer therapeutics. This Account summarizes the results of our laboratory in using a combinatorial polymer library approach to elucidate polymer structure/function relationships and enable the development of polymeric gene delivery nanoparticles with viral-like efficacy.     

Potential for Layered Double Hydroxides-Based,Innovative Drug Delivery Systems

Layered Double Hydroxides (LDHs)-based drug delivery systems have, for many years, shown great promises for the delivery of chemical therapeutics and bioactive molecules to mammalian cells in vitro and in vivo. This system offers high efficiency and drug loading density, as well as excellent protection of loaded molecules from undesired degradation. Toxicological studies have also found LDHs to be biocompatible compared with other widely used nanoparticles, such as iron oxide, silica, and single-walled carbon nanotubes. A plethora of bio-molecules have been reported to either attach to the surface of or intercalate into LDH materials through co-precipitation or anion-exchange reaction, including amino acid and peptides, ATPs, vitamins, and even polysaccharides. Recently, LDHs have been used for gene delivery of small molecular nucleic acids, such as antisense, oligonucleotides, PCR fragments, siRNA molecules or sheared genomic DNA. These nano-medicines have been applied to target cells or organs in gene therapeutic approaches. This review summarizes current progress of the development of LDHs nanoparticle drug carriers for nucleotides, anti-inflammatory, anti-cancer drugs and recent LDH application in medical research. Ground breaking studies will be highlighted and an outlook of the possible future progress proposed. It is hoped that the layered inorganic material will open up new frontier of research, leading to new nano-drugs in clinical applications.     

Inorganic nanoparticles as carriers for efficient cellular delivery

Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical properties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted.     

PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells

The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.     

Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in vitro successes that we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have used in our research to provide readers with the tools to characterize and engineer new vectors.     

Calcium Phosphate—DNA Nanocomposites: Morphological Studies and Their Bile Duct Infusion for Liver-Directed Gene Therapy

In this study, liver-directed gene transfer in rats with calcium phosphate (CaP) nanoparticles and the effect of the route of administration and surgical manipulations on transfection efficiency is reported. Formulations of CaP nanoparticles entrapping plasmid DNA (pDNA) were prepared by the reverse micellar method using two different surfactants. Transmission electron microscopy, scanning electron microscopy and dynamic light scattering were used to characterize the CaP–DNA nanocomposites. The morphological characteristics of the formulations showed a strong dependency on temperature. Gel electrophoresis experiments indicated that there was no degradation of the encapsulated pDNA, and in vitro cell transfection in HEK-293 and primary hepatocytes from rats as well as in vivo intraductal delivery experiments suggested that CaP nanoparticles led to significant and prolonged transgene expression. Therefore, our methodology gives a stable and viable formulation for hepatic gene therapy. Low-DNA dosage entrapped in CaP nanoparticles makes it an effective gene delivery system for clinical applications.     

Nanoparticles-Mediated CRISPR/Cas Gene Editing Delivery System

CRISPR/Cas system has become one of the most powerful techologies in biomedical research, and has showed great potentials in the gene related diseases. However, efficient delivery systems of CRISPR/Cas to target cells remains challenging. In recent years, nanoparticles have showned great potentials for the delivery of CRISPR/Cas systems. This paper mainly approaches the development and new strategies of CRISPR/Cas delivery systems, as well as their application in the clinical diseases. By summarizing the CRISPR/Cas systems delivery, new strategies are expected for the gene therapy.     

Intracellular Delivery of Molecular Cargo Using Cell-Penetrating Peptides and the Combination Strategies

Cell-penetrating peptides (CPPs) can cross cellular membranes in a non-toxic fashion, improving the intracellular delivery of various molecular cargos such as nanoparticles, small molecules and plasmid DNA. Because CPPs provide a safe, efficient, and non-invasive mode of transport for various cargos into cells, they have been developed as vectors for the delivery of genetic and biologic products in recent years. Most common CPPs are positively charged peptides. While delivering negatively charged molecules (e.g., nucleic acids) to target cells, the internalization efficiency of CPPs is reduced and inhibited because the cationic charges on the CPPs are neutralized through the covering of CPPs by cargos on the structure. Even under these circumstances, the CPPs can still be non-covalently complexed with the negatively charged molecules. To address this issue, combination strategies of CPPs with other typical carriers provide a promising and novel delivery system. This review summarizes the latest research work in using CPPs combined with molecular cargos including liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV) and calcium for the delivery of genetic products, especially for small interfering RNA (siRNA). This combination strategy remedies the reduced internalization efficiency caused by neutralization.     

Recent advances in nonviral vectors for gene delivery

Gene therapy has long been regarded a promising treatment for many diseases, whether acquired (such as AIDS or cancer) or inherited through a genetic disorder. A drug based on a nucleic acid, however, must be delivered to the interior of the target cell while surviving an array of biological defenses honed by evolution. Successful gene therapy is thus dependent on the development of an efficient delivery vector. Researchers have pursued two major vehicles for gene delivery: viral and nonviral (synthetic) vectors. Although viral vectors currently offer greater efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, nonviral vectors can readily transfect cells in culture, but efficient nanomedicines remain far removed from the clinic. Overcoming the obstacles associated with nonviral vectors to improve the delivery efficiency and therapeutic effect of nucleic acids is thus an active area of current research. The difficulties are manifold, including the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, and managing the targeting ability of the carriers with respect to the cells of interest. Modifying the surface with poly(ethylene glycol), that is, PEGylation, is the predominant method used to reduce the binding of plasma proteins to nonviral vectors and minimize clearance by the RES after intravenous administration. Nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors because of the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral and anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal models. Other approaches include (i) designing and synthesizing novel cationic lipids and polymers, (ii) chemically coupling the nucleic acid to peptides, targeting ligands, polymers, or environmentally sensitive moieties, and (iii) utilizing inorganic nanoparticles in nucleic acid delivery. Recently, the different classes of nonviral vectors appear to be converging, and the ability to combine features of different classes of nonviral vectors in a single strategy has emerged. With the strengths of several approaches working in concert, more hurdles associated with efficient nucleic acid delivery might therefore be overcome. In this Account, we focus on these novel nonviral vectors, which are classified as multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery, and ultrasound-responsive nucleic acid vectors. We highlight systemic delivery studies and consider the future prospects for nucleic acid delivery. A better understanding of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles should lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of a nucleic acid in this endeavor; making vectors targeted to cells with sustained release in vivo should provide an interesting research challenge.     

A supramolecular gene carrier composed of multiple cationic α-cyclodextrins threaded on a PPO-PEO-PPO triblock polymer

Cationic polymers have been studied as promising nonviral gene delivery vectors. In contrast to the conventional polycations with long sequences of covalently bonded repeating units, this work reports a supramolecular gene carrier where many cationic cyclic units are threaded over a polymer chain to form a chain-interlock structured gene carrier. A series of novel supramolecular cationic polyrotaxanes consisting of multiple α-cyclodextrin (α-CD) rings grafted with various linear or nonlinear oligoethylenimine (OEI) chains, which are threaded and capped over a reverse Pluronic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEO-PPO) amphiphilic triblock copolymer chain, were synthesized and characterized in term of their molecular and supramolecular structures, DNA binding and condensation ability, cytotoxicity, and in vitro gene transfection efficiency in cultured cells. The supramolecular cationic polyrotaxanes were found to contain 8 cationic α-CD rings that are threaded on a PPO-PEO-PPO triblock copolymer chain. They demonstrated strong ability to bind and condense plasmid DNA into nano-sized particles which are suitable for gene delivery. In both HEK293 and COS7 cells, these polyrotaxanes show low cytotoxicity and high transfection efficiency. In particular, the cationic polyrotaxanes displayed sustained gene delivery capability in HEK293 cells in both serum and serum free condition with the increasing expression duration.     

Generation of carrier peptides for the delivery of nucleic acid drugs in primary cells

Now that the human genome has been decoded, the demand for novel therapeutic concepts, such as gene and stem cell therapy, is higher than ever before. Although new and better pharmaceutical agents are available, their efficient delivery to the intracellular site of action is still a serious challenge. A possible solution to this problem is the use of cell-penetrating peptides as delivery vectors, including derivatives of human calcitonin (hCT). The aim of this study was to synthesise novel branched hCT-derived peptides for the noncovalent delivery of nucleic acids. The uptake of the resulting oligocationic peptides into various cell lines as well as primary cells was monitored by fluorescence microscopy. To determine the appropriate peptide-plasmid charge ratios for efficient cell transfection, electromobility shift assays were carried out. Finally, flow cytometric and fluorescence microscopic studies of gene expression highlighted two novel hCT-derived peptides as highly effective in the delivery of noncovalently complexed plasmid DNA. Thus, the absence of cytotoxicity paired with highly efficient cell internalisation and transfection rates, in primary cells as well, make both peptides powerful candidates as drug delivery vectors, especially for plasmid DNA, for both in vivo and ex vivo therapeutic applications.     

Nanoparticle popsicle: Transdermal delivery of nanoparticles using polymeric microneedle array

A number of nanoparticles are currently applied for medical or medicinal research fields through conjugating with drugs or bio-molecules such as DNA, RNA, and peptides. There are three distinct ways to deliver nanoparticles into animal and human bodies: injection, feeding, and transdermal administration. We fabricated a polymeric microneedle array carrying nanoparticles on the end-tip of needles for an efficient delivery of functional nanoparticles through the skin. The polymeric microneedles with a length ranging from 600 to 1,000 μm were casted out using laser-printed PDMS (polydimethylsiloxane) molds where the 50 nm silica or polystyrene nanoparticles had been filled by centrifugation. When the microneedle array was applied to the porcine cadaver skin, nanoparticles were quickly and stably dispersed in the hypodermis. Microneedle array conjugated with nanoparticles has a potential for an alternative method to deliver cosmetic or pharmaceutical materials into human skin locally without professional procedures.     

A sight on the current nanoparticle-based gene delivery vectors

Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/in vivo transformation efficiency.     

Chloroquine-enhanced gene delivery mediated by carbon nanotubes

Polyethyleneimine-coated double-walled carbon nanotubes (DWCNTs) were used for dual gene and drug delivery, after loading the DWCNTs with the drug chloroquine, a lysosomotropic compound that is able to promote escape from the lysosomal compartment. Different forms of functionalization of the DWCNTs were examined in order to optimize this system. They included the testing of different treatments on DWCNTs to optimize the loading and delivery of chloroquine and the selection of a cationic polymer for coating the DWCNTs for optimum DNA binding and delivery. An acid oxidation treatment of DWCNTs was selected for optimum chloroquine loading together with polyethyleneimine as optimum cationic coating agent for plasmid DNA binding. Optimization of the conditions for choroquine-enhanced gene delivery were developed using luciferase expression as a model system. We have demonstrated that chloroquine-loading increases the ability of polyethyleneimine-coated DWCNTs to deliver functional nucleic acid to human cells. Cell viability tests have shown no cytotoxicity of the functionalized DWCNTs at the concentrations needed for optimum gene delivery. These results support the potential applications of this methodology in gene therapy.     

Characteristics of functionalized nano-hydroxyapatite and internalization by human epithelial cell

Hydroxyapatite is the main inorganic component of biological bone and tooth enamel, and synthetic hydroxyapatite has been widely used as biomaterials. In this study, a facile method has been developed for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). The synthesized nanoparticles characterized by transmission electron microscopy, X-ray diffractometry, Fourier transform infrared, and Zeta potential analyzer. Its biological properties with DNA binding, cell toxicity, cell binding and intracellular distribution were tested by agarose gel electrophoresis assay, flow cytometry, and fluorescence microscope and laser scanning confocal microscope. The synthesized Arg-Eu-HAP could effectively bind DNA without any cytotoxicity and be internalized into the cytoplasm and perinuclear of human lung epithelial cells.     

Redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles for controllable gene delivery and magnetic resonance imaging

Recently, theranostic candidates that provide a combination of gene delivery and image diagnosis have attracted much interest in medical research. However, there are still many challenges for their clinical applications, such as uncontrollable gene delivery, high cytotoxicity, low transfection efficiency and reduced image contrast. Herein, redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles (IONs@rPEI) were prepared for both efficient gene delivery and magnetic resonance (MR) imaging. Firstly, crosslinked rPEI was synthesized by Michael addition reaction with N,N‐bis(acryloyl)cystamine, dopamine and low‐molecular‐weight branched PEI. The rPEI was then coated onto IONs by ligand exchange reaction forming IONs@rPEI. The physicochemical properties of the IONs@rPEI, such as chemical structure, size, zeta potential and DNA condensation ability, were investigated. In addition, a rapid degradation of the as‐prepared nanoparticles was observed, which was triggered by reducing glutathione via destruction of disulfide linkages suggesting a potential controllable DNA release in tumor cells. In MR imaging detection, the IONs@rPEI had a high T₂ relaxivity of 81 L mmol^?1 s^?1 indicating a potential usage as MR imaging contrast reagent. In cell assay, the IONs@rPEI exhibited low cytotoxicity and good transfection efficiency. In conclusion, the as‐prepared crosslinked IONs@rPEI can be used as a promising technology platform for gene therapy and MR imaging in theranostics. © 2019 Society of Chemical Industry     

阳离子PLGA纳米颗粒作为核酸疫苗递送载体的能力及其特性评价

目的对阳离子聚乳酸-羟基乙酸共聚物[poly(D,L-lactide-co-glycolide),PLGA]纳米颗粒作为核酸疫苗载体的能力及其特性进行评价。方法采用双重乳化法制备阳离子PLGA纳米颗粒,检测其粒径分布和表面电势,获得合成的最适方案。通过考察其形貌、吸附效率、抵抗核酸酶降解能力、细胞毒性、细胞摄取及转染真核表达质粒的能力,分析其作为核酸疫苗载体的适用性及可行性。结果由两种试剂修饰PLGA制备的纳米颗粒粒径分布、表面电势及分散性最佳;透射电镜及扫描电镜下观察粒径均一且呈规则球形;对质粒DNA的吸附效率可达65%;能够有效保护吸附于纳米颗粒的质粒DNA免受核酸酶降解;两种细胞系中的细胞毒性试验均显示细胞存活率高于80%;共聚焦显微镜下可观察到其能被细胞内化;间接免疫荧光试验结果表明,其能有效转染真核表达质粒并使其正确表达。结论成功制备了具有递送核酸疫苗能力的阳离子PLGA纳米颗粒,为核酸疫苗载体的发展奠定了理论基础。