Decoding Cosmetic Complexities: A Comprehensive Guide to Matrix Composition and Pretreatment Technology.

Despite advancements in analytical technologies, the complex nature of cosmetic matrices, coupled with the presence of diverse and trace unauthorized additives, hinders the application of these technologies in cosmetics analysis. This not only impedes effective regulation of cosmetics but also leads to the continual infiltration of illegal products into the market, posing serious health risks to consumers. The establishment of cosmetic regulations is often based on extensive scientific experiments, resulting in a certain degree of latency. Therefore, timely advancement in laboratory research is crucial to ensure the timely update and adaptability of regulations. A comprehensive understanding of the composition of cosmetic matrices and their pretreatment technologies is vital for enhancing the efficiency and accuracy of cosmetic detection. Drawing upon the China National Medical Products Administration's 2021 Cosmetic Classification Rules and Classification Catalogue, we streamline the wide array of cosmetics into four principal categories based on the following compositions: emulsified, liquid, powdered, and wax-based cosmetics. In this review, the characteristics, compositional elements, and physicochemical properties inherent to each category, as well as an extensive overview of the evolution of pretreatment methods for different categories, will be explored. Our objective is to provide a clear and comprehensive guide, equipping researchers with profound insights into the core compositions and pretreatment methods of cosmetics, which will in turn advance cosmetic analysis and improve detection and regulatory approaches in the industry.

Quantification of sesquiterpene pyridine alkaloids from genus Tripterygium by band-selective HSQC NMR.

Sesquiterpene pyridine alkaloids (SPAs) are bioactive analogues derived from the genus Tripterygium and have anti-inflammatory and anti-rheumatic properties. Attributed to the similar sesquiterpene structures, the total SPAs showed severe peak overlap in 1D NMR and HPLC, leading to difficulties in identification and quantification. Interestingly, the application of band-selective HSQC NMR that specifically excited the region corresponding to the H-3 of SPAs prompted a signal separation of the total SPAs. Based on the high resolution, 23 SPAs were identified from the band-selective HSQC spectrum. The coupling constants (JCH, JHH) and relaxation times (T1, T2) of SPAs were measured, and it was found that they caused less than 1% attenuation of the HSQC signals, so the HSQC signals of SPAs had almost uniform responses. The concentrations of 23 SPAs were determined by standard curve method, using wilforgine as the calibration. In addition, we extended the pulse length-based concentration determination (PULCON) as a more efficient external standard method to the band-selective HSQC spectrum, and the results showed that the concentrations of alkaloids determined by PULCON were consistent with those measured by standard curve method. The developed quantification approach was validated according to the <761> of United States Pharmacopoeia (USP), demonstrating that the established band-selective HSQC approach is reliable for the rapid quantification of analogues in botanical extracts.

Screening, characterization, and determination of suspected additives bimatoprost and latanoprost in cosmetics using NMR and LC-MS methods.

Recently, many new types of cosmetic illegal additives have been screened in the market. Most of the new additives were new drugs or analogues with very similar structures to other prohibited additives, which were difficult to be identified by liquid chromatography-mass spectrometry (LC-MS) only. Therefore, a new strategy is proposed, which is chromatographic separation combined with nuclear magnetic resonance spectroscopy (NMR) structural identification. The suspected samples were screened by ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UPLC-Q-TOF-MS), followed by purification and extraction through silica-gel column chromatography and preparative high-performance liquid chromatography (HPLC). Finally, the extracts were identified unambiguously by NMR as bimatoprost and latanoprost, which were identified to be new cosmetic illegal additives in eyelash serums in China. Meanwhile, bimatoprost and latanoprost were quantified by high-performance liquid chromatography tandem triple quadrupole mass spectrum (HPLC-QQQ-MS/MS). The quantitative method demonstrated good linearity in the range of approximately 0.25-50 ng/mL (R2 > 0.9992), with limit of detection (LOD) and limit of quantification (LOQ) values of 0.01 and 0.03 mg/kg, respectively. The accuracy, precision, and reproducibility were confirmed to be acceptable.

Identification and characterization of forced degradation products of 5-hydroxymethyl-2-furaldehyde (5-HMF) by HPLC, LC-LTQ/Orbitrap and NMR studies.

5-Hydroxymethyl-2-furaldehyde (5-HMF) is a kind of aldehyde compound with highly active furan ring, which is generated by dehydration of glucose, fructose, and other monosaccharides. It widely exists in drugs, foods, health products, cosmetics, and traditional Chinese medicine preparations with high sugar content. Due to the toxicity, the concentration of 5-HMF was always monitored to identify non-conformities and adulteration, as well as ensure the process efficiency, traceability and safety in foods or drugs in the pharmacopoeias of various countries. Herein, a comprehensive forced degradation study was performed to characterize the degradation products (DPs) of 5-HMF under hydrolytic (neutral, acidic, and alkaline) degradation, oxidative, thermal, humidity, and photolytic degradation conditions. A total of five degradants were identified, and two of them (DP-3 and DP-5) were novel DPs first reported in our study. Major DPs (i.e., DP-1 and DP-2) with relatively high peak areas were isolated using semi-preparative HPLC and characterized by LC-LTQ/Orbitrap and NMR. 5-HMF was only stable in alkaline hydrolysis condition. In addition, the degradation pathways and mechanism of these DPs were also explained using LC-LTQ/Orbitrap. In silico toxicity and metabolism behavior of the DPs were evaluated using Derek Nexus and Meteor Nexus software, respectively. The predicted toxicity data indicated that both the drug 5-HMF and its DPs bear the potential of hepatotoxicity, mutagenicity, chromosome damage, and skin sensitisation. Our research may be beneficial for the quality control and suitable storage conditions of 5-HMF.

Synthesis and antibacterial activity of novel 2­fluoro ketolide antibiotics with 11,12­quinoylalkyl side chains.

A series of novel 2­fluoro ketolide antibiotics with 11,12­quinoylalkyl side chains derived from telithromycin and cethromycin were designed and synthesized. The corresponding targets 2a-o were tested for their in vitro activities against a series of macrolide-sensitive and macrolide-resistant pathogens. Some of them showed a similar antibacterial spectrum and comparable or slightly better activity to telithromycin. Among them, compounds 2g and 2k, displayed excellent activities against macrolide-sensitive and macrolide-resistant pathogens.

Immunosuppressive Sesquiterpene Pyridine Alkaloids from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A-J (1-10), were isolated from the roots of T. wilfordii, along with ten known analogues (11-20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9-16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC50 values of 7.25 µg/mL, 8.75 µM, 0.74 µM, and 15.66 µM, respectively, and no influence on the cell viability at a concentration of 100 µg/mL (TA) or 100 µM (5, 11, and 16). Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.

[Spectral characteristics of sesquiterpene pyridine alkaloids from Tripterygium plants].

Sesquiterpene pyridine alkaloids are important components in Tripterygium plants, possessing a wide range of pharmacological activities, such as anti-inflammation immunosuppression, anti-tumor, anti-virus, and deinsectization, and are of great research value. They are composed of highly oxidized dihydro-ß-furansquiterpene and pyridine dicarboxylic acid through ester bonds. According to the structural characteristics of pyridine dicarboxylic acid fragments, they can be divided into various structural subtypes. Up to now, more than 110 sesquiterpene pyridine alkaloids have been isolated and identified from Tripterygium plants. This study reviewed the structural features and spectral(i.e., UV, IR, MS, and NMR) characteristics of sesquiterpene pyridine alkaloids and summarized the structural elucidation process in detail to provide references for their further research and development.

Comprehensive Evaluation of the Quality of Tripterygium Glycosides Tablets Based on Multi-Component Quantification Combined with an In Vitro Biological Assay.

Tripterygium glycosides tablets (TGTs) are widely used in clinical practice to treat rheumatoid arthritis and other autoimmune diseases, with significant beneficial effects but also high toxicity, necessitating rigorous quality evaluation and control. In current study, a rapid resolution liquid chromatography tandem electrospray ionization triple quadrupole mass spectrometry (RRLC-ESI-MS/MS) method was developed and validated for the quantitative analysis of 14 components of ten batches of TGTs produced by different manufacturers, including four diterpenoids, three triterpenoids, and seven sesquiterpene alkaloids. Meanwhile, the NO inhibition effects of these TGTs were evaluated in LPS-induced RAW264.7 cells for their downstream anti-inflammatory activities, as well as their cytotoxicity. The results indicate that the TGTs from different manufacturers showed poor quality consistency, as evidenced by large variations in chemical profiles and biological effects, which may increase the risks associated with clinical use. To improve the quality status of TGTs, it is crucial to identify indicator components whose characterization can accurately reflect the efficacy and toxicity of TGTs from which they were derived. Our study reveals that triptolide, triptoquinone B, celastrol, and demethylzelaysteral considerably contributed to the anti-inflammatory activity and/or cytotoxicity of TGTs, implying that they should be further investigated as candidate indicator components for TGT quality control.

Characterization and determination of benvitimod, an unknown risk substance in cosmetics, using nuclear magnetic resonance spectroscopy and HPLC-MS/MS.

Ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry, combined with preparative chromatography and nuclear magnetic resonance spectroscopy, a new method for identifying unknown risk substance structures in cosmetics has been established. Moreover, HPLC-MS/MS was developed for the determination of benvitimod in cosmetics. The sample was collected in ultrahigh-performance liquid chromatography-tandem high-resolution mass spectrometry, and the molecular formula of the unknown was obtained as C17 H18 O2 . After preparative chromatography enrichment and purification, the enriched compound was scanned by nuclear magnetic resonance spectroscopy, and the chemical structure of the unknown compound was confirmed to be benvitimod. Subsequently, the separation was determined in multiple reaction monitoring mode. The results showed that the linearity of benvitimod was good in the range of 1-100 µg/L with a correlation coefficient r2  > 0.999; the limits of detection and quantification were 0.02 mg/kg and 0.067 mg/kg; the precision and stability were good; and the average recoveries were 104.2%, 108.2%, and 108.7% for low, medium, and high spiked concentrations, respectively. Forty batches of cosmetics were screened, of which two batches were detected with the illegal addition of benvitimod at 2.48 and 3.13 g/kg. The method effectively solved the loopholes in regulation and provided a research basis for the qualitative identification of structurally unknown compounds in cosmetics.

Quality control and product differentiation of LMWHs marketed in China using 1H NMR spectroscopy and chemometric tools.

Low molecular weight heparins (LMWHs) are heterogeneous mixtures of glycosaminoglycan chains composed of mixture of different lengths and substitution patterns. Structural characterization and quality control of LMWHs have always been challenging. The Chinese drug regulatory authorities have been committed to improve the supervision standards of LMWHs to better regulate the quality and safety of LMWHs in current Chinese market. In the present paper, 80 batches of three types LMWHs (dalteparin, enoxaparin and naldroparin) marketed in China from different manufacturers were studied by 1H NMR experiments and chemometric analysis. The method can be used not only to monitor impurities and contaminants, but also to check the batch-to-batch consistency of each manufacture. Moreover, for the biosimilar LMWHs from different manufactures, they can be differentiated and clustered according to their slightly different structural compositions originated from production process. By using this method, the quality and safety of LMWHs marketed in China were initially assessed.

Laser-assisted skin delivery of immunocontraceptive rabies nanoparticulate vaccine in poloxamer gel.

A painless skin delivery of vaccine for disease prevention is of great advantage in improving compliance in patients. To test this idea as a proof of concept, we utilized a pDNA vaccine construct, pDNAg333-2GnRH that has a dual function of controlling rabies and inducing immunocontraception in animals. The pDNA was administered to mice in a nanoparticulate form delivered through the skin using the P.L.E.A.S.E.® (Precise Laser Epidermal System) microporation laser device. Laser application was well tolerated, and mild skin reaction was healed completely in 8 days. We demonstrated that adjuvanted nanoparticulate pDNA vaccine significantly upregulated the expression of co-stimulatory molecules in dendritic cells. After topical administration of the adjuvanted nano-vaccine in mice, the high avidity serum for GnRH antibodies were induced and maintained up to 9 weeks. The induced immune response was of a mixed Th1/Th2 profile as measured by IgG subclasses (IgG2a and IgG1) and cytokine levels (IFN-γ and IL-4). Using flow cytometry, we revealed an increase of CD8+ T-cells and CD45R B cells upon the administration of the adjuvanted vaccine. Our previous study used the same pDNA nanoparticulate vaccine through an IM route, and a comparable immune response was induced using P.L.E.A.S.E. However, the vaccine dose in the current study was four-fold less than what was applied through the IM route.We concluded that laser-assisted skin vaccination has a potential of becoming a safe and reliable vaccination tool for rabies vaccination in animals or even in humans for pre- or post-exposure prophylaxis.

SAFETY, IMMUNOGENICITY, AND EFFICACY OF INTRAMUSCULAR AND ORAL DELIVERY OF ERA-G333 RECOMBINANT RABIES VIRUS VACCINE TO BIG BROWN BATS (EPTESICUS FUSCUS).

Attenuated strains of rabies virus (RABV) have been used for oral vaccination of wild carnivores in Europe and North America. However, some RABV vaccines caused clinical rabies in target animals. To improve the safety of attenuated RABV as an oral vaccine for field use, strategies using selection of escape mutants under monoclonal antibody neutralization pressure and reverse genetics-defined mutations have been used. We tested the safety, immunogenicity, and efficacy of one RABV construct, ERA-g333, developed with reverse genetics by intramuscular (IM) or oral (PO) routes in big brown bats (Eptesicus fuscus). Twenty-five bats received 5×106 mouse intracerebral median lethal doses (MICLD50) of ERA-g333 by IM route, 10 received 5×106 MICLD50 of ERA-g333 by PO route, and 22 bats served as unvaccinated controls. Twenty-one days after vaccination, 44 bats were infected by IM route with 102.9 MICLD50 of E. fuscus RABV. We report both the immunogenicity and efficacy of ERA-g333 delivered by the IM route; no induction of humoral immunity was detected in bats vaccinated by the PO route. Two subsets of bats vaccinated IM (n=5) and PO (n=3) were not challenged, and none developed clinical rabies from ERA-g333. Scarce reports exist on the evaluation of oral rabies vaccines in insectivorous bats, although the strategy evaluated here may be feasible for future application to these important RABV reservoirs.

[Research progress on chemical constituents and quality control of Tripterygium wilfordii preparations].

Tripterygium wilfordii preparations,with various biological activities such as immunosuppressive,anti-inflammatory and anti-cancer effects,are widely used in the treatment of autoimmune diseases such as rheumatoid arthritis,lupus erythematosus,and nephrotic syndrome. They have definite therapeutic effect,but often cause serious adverse reactions and result in damages to liver,kidney,blood,reproduction,and other systems due to their complex compositions,great toxicity,and narrow margin between the toxic and therapeutic dosages. At present,T. wilfordii preparations produced by different manufacturers exhibit large variations in clinical efficacy and side effects in account of their different chemical compositions and quality fluctuation due to differences in raw materials and production process. However,the existing quality standards are controversial in terms of index components and content limit,which cannot be effectively used for the overall quality control of the preparations. In this paper,the research progress on chemical constituents,quality standard and quality control methods of four T. wilfordii preparations including Tripterygium Tablets,Tripterygium Zongtie Tablets,Tripterygium Shuangceng Tablets and Tripterygium Glycosides Tablets was reviewed,in order to provide ideas and reference for the quality improvement of this type of preparations.

Evaluation of an adjuvanted hydrogel-based pDNA nanoparticulate vaccine for rabies prevention and immunocontraception.

Immunocontraceptive vaccination is becoming an acceptable strategy in managing animal populations. Mass vaccination of dogs is the most cost-effective and efficient method to control rabies, and combination of rabies vaccination and animal population control will be an added advantage. In this study, we developed an adjuvanted hydrogel-based pDNA nanoparticulate vaccine for rabies protection and immunocontraception. In vivo, we observed an immune response skewed toward a Th2 type, in contrast to the Th1 type in our previous pDNA study. The observation was verified by the IgG2a/IgG1 ratio (<1), and cytokine expression profile of IL-4 and IFN-γ. The humoral immune response is key for rabies protection and a GnRH antibody-based immunocontraception. In mice, anti-GnRH antibody titers were detected 4 weeks after immunization and lasted for 12 weeks, post animal experiment was terminated. The adjuvanted pDNA nanoparticulate vaccine shows promise for future studies evaluating protection from rabies challenge and prevention of animal breeding.

Inactivated Rabies Virus-Vectored Immunocontraceptive Vaccine in a Thermo-Responsive Hydrogel Induces High and Persistent Antibodies against Rabies, but Insufficient Antibodies against Gonadotropin-Releasing Hormone for Contraception.

Rabies is preventable through vaccination, but the need to mount annual canine vaccination campaigns presents major challenges in rabies control and prevention. The development of a rabies vaccine that ensures lifelong immunity and animal population management in one dose could be extremely advantageous. A nonsurgical alternative to spay/neuter is a high priority for animal welfare, but irreversible infertility in one dose has not been achieved. Towards this goal, we developed a rabies virus-vectored immunocontraceptive vaccine ERA-2GnRH, which protected against rabies virus challenge and induced >80% infertility in mice after three doses in a live, liquid-vaccine formulation (Wu et al., 2014). To improve safety and use, we formulated an inactivated vaccine in a thermo-responsive chitosan hydrogel for one-dose delivery and studied the immune responses in mice. The hydrogel did not cause any injection site reactions, and the killed ERA-2GnRH vaccine induced high and persistent rabies virus neutralizing antibodies (rVNA) in mice. The rVNA in the hydrogel group reached an average of 327.40 IU/mL, more than 200 times higher than the liquid vaccine alone. The Gonadotropin-releasing hormone (GnRH) antibodies were also present and lasted longer in the hydrogel group, but did not prevent fertility in mice, reflecting a possible threshold level of GnRH antibodies for contraception. In conclusion, the hydrogel facilitated a high and long-lasting immunity, and ERA-2GnRH is a promising dual vaccine candidate. Future studies will focus on rabies protection in target species and improving the anti-GnRH response.

Feline herpesvirus vectored-rabies vaccine in cats: A dual protection.

In China, cats cause about 5% of human rabies cases. Rabies control in cats plays a role in achieving the ultimate goal of elimination of dog rabies-mediated human deaths. However, there is no cat-specific rabies vaccine in China yet. In this study, we constructed a recombinant rabies vaccine by using a felid herpesvirus 1 (FHV-1) isolate, and deleted the gI/E in the FHV-1 and replaced the region with a glycoprotein (G) of rabies virus (RABV) strain BD06 through homologous recombination. The recombinant virus FHV-RVG was recovered and purified, and the expression of RABV glycoprotein was verified by indirect immunofluorescent assay. For potency in cats, each animal was inoculated intranasally with 1 ml FHV-RVG at 106.5 TCID50. Blood samples were collected at defined intervals for antibody titration. The animals were challenged by herpes and rabies after completion of vaccination on day 180 and day 194, respectively. Our results demonstrated all vaccinated cats generated antibodies against both FHV-1 and RABV, and reached an arbitrary protective titer > 0.5 IU/ml for rabies viral neutralizing antibody (VNA) by day 14 post inoculation (dpi) and titer peaked on 30 dpi with VNA at 24.5 ±â€¯10.23 IU/ml. All vaccinated cats presented no clinical signs of FHV-1 infection and survived rabies challenge, while the control cats had severe rhinotracheitis and died from rabies after challenge. All this demonstrated that the FHV-based recombinant vaccine is effective in protection against both FHV-1 and RABV infections.

Novel mass spectrometry based detection and identification of variants of rabies virus nucleoprotein in infected brain tissues.

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms.

A high throughput neutralization test based on GFP expression by recombinant rabies virus.

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers.

Development of a relative potency test using ELISA for human rabies vaccines.

The NIH potency test for human rabies vaccines has disadvantages for use, especially in developing countries where rabies is endemic and prophylaxis needs ample, rapid, and reliable vaccine supplies. In China, 60-75 million doses of human rabies vaccines are administered each year. Vaccine quality control is of paramount importance, as is the release of potency-validated vaccines. We intended to design an alternative to the NIH in vivo method, and developed a relative potency test using an ELISA. Using Pearson's correlation analysis, we found a close relationship between the rabies vaccine glycoprotein content in vitro and the potency values in vivo. We suggest the relative potency test developed here as a simplified method for human rabies vaccine quality control in China and a possible alternative to the NIH method.

Characterization of rabies pDNA nanoparticulate vaccine in poloxamer 407 gel.

Plasmid DNA (pDNA) vaccines have the potential for protection against a wide range of diseases including rabies but are rapid in degradation and poor in uptake by antigen-presenting cells. To overcome the limitations, we fabricated a pDNA nanoparticulate vaccine. The negatively charged pDNA was adsorbed onto the surface of cationic PLGA (poly (d, l-lactide-co-glycolide))-chitosan nanoparticles and were used as a delivery vehicle. To create a hydrogel for sustainable vaccine release, we dispersed the pDNA nanoparticles in poloxamer 407 gel which is liquid at 4 °C and turns into soft gels at 37 °C, providing ease of administration and preventing burst release of pDNA. Complete immobilization of pDNA to cationic nanoparticles was achieved at a pDNA to nanoparticles ratio (P/N) of 1/50. Cellular uptake of nanoparticles was both time and concentration dependent and followed a saturation kinetics with Vmax of 11.389 µg/mL h and Km of 139.48 µg/mL. The in vitro release studies showed the nanoparticulate vaccine has a sustained release for up to 24 days. In summary, pDNA PLGA-chitosan nanoparticles were non-cytotoxic, their buffering capacity and cell uptake were enhanced, and sustained the release of pDNA. We expect our pDNA vaccine's potency will be greatly improved in the animal studies.