The effects of Staphylococcus aureus protein a (SpA) on the expression of inflammatory cytokines in autoimmune patients and their probable immune response modulation mechanisms.

The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1ß, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.

Preparation of graft copolymer of chitosan-poly ortho-toluidine for antibacterial properties.

The combination polymers or copolymers have new and combined properties and increase the efficiency of the new polymer. Biopolymers are biodegradable and can play the role of biocompatible and biodegradable in composite polymers. Therefore, poly ortho-toluidine was grafted on chitosan (Cs-g-POT) by chemical and electrochemical polymerization methods. Cs-g-POT was characterized by FTIR, UV-visible, and 1H NMR spectroscopy techniques. The thermal behaviors of the copolymer were investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The images of the surface of the copolymer obtained from imaging SEM confirm the successful attachment of POT on chitosan and indicate that the graft polymerization has been successfully performed with both methods. The percentage and efficiency of engraftment were carefully measured and reported. The electrical conductivity of Cs-g-POT was measured by the four-point method and the conductivity was 9.1 × 10-4 S/cm. The copolymer's antibacterial property was studied on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa as a common bacterium in skin wounds. These studies were investigated using the disk diffusion and minimum inhibitory concentration (MIC) methods. In all tested concentrations the polymer could inhibit the growth of E. coli and P. aeruginosa significantly. However, it inhibited the growth of S. aureus in concentrations above 1 µg. Bacteria are adsorbed on the surface of the polymer by polar-polar and Van Der Waals interactions, where they undergo cell lysis by dopant and electron transfer, and eventually bacterial cell death. Due to its scaffolding properties, this polymer will have a very good use in tissue and bone repair as well as anti-cancer drugs.

Nano and microparticle drug delivery systems for the treatment of Brucella infections.

Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients.

Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection.

Viral hepatitis, the most common cause of inflammatory liver disease, affects hundreds of millions of people worldwide. It is most commonly associated with one of the five nominal hepatitis viruses (hepatitis A-E viruses). HBV and HCV can cause acute infections and lifelong, persistent chronic infections, while HAV and HEV cause self-limiting acute infections. HAV and HEV are predominantly transmitted through the fecal-oral route, while diseases transmitted by the other forms are blood-borne diseases. Despite the success in the treatment of viral hepatitis and the development of HAV and HBV vaccines, there is still no accurate diagnosis at the genetic level for these diseases. Timely diagnosis of viral hepatitis is a prerequisite for efficient therapeutic intervention. Due to the specificity and sensitivity of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated sequences (Cas) technology, it has the potential to meet critical needs in the field of diagnosis of viral diseases and can be used in versatile point-of-care (POC) diagnostic applications to detect viruses with both DNA and RNA genomes. In this review, we discuss recent advances in CRISPR-Cas diagnostics tools and assess their potential and prospects in rapid and effective strategies for the diagnosis and control of viral hepatitis infection.

Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination.

BACKGROUND: Targeted gene inactivation (TGI) is a widely used technique for the study of genes' functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR). METHODS: For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing. RESULTS: The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted. CONCLUSION: In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria's genes.

Immunological detection of AcAMP antimicrobial peptide secreted by Aspergillus clavatus.

BACKGROUND AND OBJECTIVES: Aspergillus clavatus antimicrobial peptide (AcAMP) is a fungi-derived peptide with a broad spectrum of activity against pathogenic bacteria and fungi. Natural antimicrobial peptides, including AcAMP, have attracted many attentions in the development of new natural antibiotics against pathogenic bacteria, especially multidrug resistant ones. MATERIALS AND METHODS: In the present study, acamp gene was codon-optimized and chemically synthesized in pUC57 cloning vector, subcloned into pET28a (+) expression vector and transferred into competent Escherichia coli BL21 (DE3) cells. The expression of AcAMP was induced by addition of Isopropyl ß- d-1-thiogalactopyranoside (IPTG) and the expressed peptide was purified by Ni-NTA. BALB/c mice were immunized with the purified peptide and the ability of the immunized mice sera for the detection of the native AcAMP secreted by A. clavatus IRAN 142C was examined through ELISA and Western blotting techniques. RESULTS: Both ELISA and Western blotting demonstrated the ability of the sera of the immunized mice to detect the native AcAMP. CONCLUSION: The results of the present work show that the raised antibody against recombinant AcAMP can be used to detect AcAMP peptide, an issue which paves the way to develop detection kits for the detection of AcAMP-producing organisms, purification of this valuable peptide for further investigations.

Nanotechnology as a promising platform for rheumatoid arthritis management: Diagnosis, treatment, and treatment monitoring.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that develops in about 5 per 1000 people. Over the past years, substantial progresses in knowledge of the disease's pathophysiology, effective diagnosis methods, early detection, and efficient treatment strategies have been made. Notably, nanotechnology has emerged as a game-changer in the efficacious management of many diseases, especially for RA. Joint replacement, photothermal therapy (PTT), photodynamic therapy (PDT), RA diagnosis, and treatment monitoring are nano-based avenues in RA management. Here, we present a brief overview of the pathogenesis of RA, risk factors, conventional diagnostic methods and treatment approaches, and then discuss the role of nanomedicine in RA diagnosis, treatment, and treatment monitoring with an emphasis on functional characteristics distinctive from other RA therapeutics.

Mesoporous silica nanoparticles-based formulations of a chimeric proteinous vaccine candidate against necrotic enteritis disease.

To develop a nanoparticle-based vaccine against necrotic enteritis, a chimeric antigen (rNA) consisting of the main antigens of Clostridium perfringens, NetB, and Alpha toxin, was prepared. Then, the rNA molecules were loaded onto the functionalized mesoporous silica nanoparticles (MSNPs) using physical adsorption or covalent conjugation methods. The characterization of synthesized nanoparticles was performed by scanning electron microscopy, dynamic light scattering, zeta potential measurement, Fourier transform infrared spectroscopy, and thermogravimetry techniques. The results revealed that the spherical nanoparticles with an average diameter of 90 ±â€¯12 nm and suitable surface chemistries are prepared. MSNPs-based formulations did not show any significant toxicity on the chicken embryo fibroblast cells. The results of the challenge experiments using subcutaneous or oral administration of the as-prepared formulations in the animal model showed that the as-prepared nanosystems, similar to those formulated with a commercial adjuvant (Montanide), present stronger humoral immune responses as compared to that of the free proteins. It was also indicated that the best protection is obtained in groups vaccinated with MSNPs-based nanovaccine, especially those who orally received covalently conjugated nanovaccine candidates. These results recommend that the MSNPs-based formulated chimeric proteinous vaccine candidates can be considered as an effective immunizing system for the oral vaccination of poultry against gastrointestinal infectious diseases.

A meta-analysis of the efficiency of metal nanoparticles in vaccine delivery against infectious disease.

Background: Exploration of the efficiency of metal nanoparticles as adjuvants have reported varying results. Objective: The efficacy of metal nanoparticles as adjuvants was investigated Data sources: Database were searched using the terms 'metal nanoparticles' and 'vaccines'. Study eligibility criteria: Studies in animal models utilizing any metal-based vaccines, where the survival rate was described. Study appraisal: The quality of the studies was examined using aspects of the ARRIVE guidelines and assessment of the risk of bias of included studies. Results: Metal nanoparticle-based adjuvants were more effective compared with control (unvaccinated groups) but have not been more successful in competing with common adjuvants or even antigens alone. Limitation: More than 75% of articles have used only gold nanoparticles. Conclusion: Nano-adjuvants do not have a significant effect on reducing mortality.

Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers.

Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this surface display system that need to be addressed in the future. VLPs that are classified as subunit vaccines are subdivided into enveloped and non- enveloped subtypes both of which are discussed in this review article. VLPs have also recently received attention for their successful applications in targeted drug delivery and for use in gene therapy. The development of more effective and targeted forms of VLP by modification of the surface of the particles in such a way that they can be introduced into specific cells or tissues or increase their half-life in the host is likely to expand their use in the future. Recent advances in the production and fabrication of VLPs including the exploration of different types of expression systems for their development, as well as their applications as vaccines in the prevention of infectious diseases and cancers resulting from their interaction with, and mechanism of activation of, the humoral and cellular immune systems are discussed in this review.

Correction to: CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.

An amendment to this paper has been published and can be accessed via the original article.

CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.

Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.

Immunogenicity Evaluation of Recombinant Staphylococcus aureus Enterotoxin B (rSEB) and rSEB-loaded Chitosan Nanoparticles Following Nasal Administration.

Staphylococcal enterotoxin B (SEB), apotent superantigen, is responsible for many disorders caused by Staphylococcus aureus. With regard to the appearance of multidrug-resistant strains of the bacteria, there is a great need to develop an efficient vaccine against this pathogen. In the present study, the immunogenicity of recombinant SEB was evaluated following nasal administration to BABLB/c mice. Indeed, the rSEB protein was entrapped into chitosan nanoparticles and the immunogenicity of nano-formulation was investigated. SEB protein was expressed in E. coli BL21 (DE3) and purified by using a nickel column. Chitosan nanoparticles were synthesized in the presence of rSEB; using the ionic gelation technique. Synthesized NPs containing rSEB and bare rSEB were administered to mice nasally. Serum and stool IgG and IgA antibody showed that both formulations were able to evoke the mice's immune responses and there was no significant difference between them. Results of the toxin neutralization test on Vero cells indicated that the sera of the immunized mice had an inhibitory effect on the growth of these cells (p<0.001). Nasal administration of bare rSEB could efficiently simulate the mice's immune system and nano-delivery of this protein via nasal route had not a significant impact on its immunogenicity improvement.

Production of egg yolk antibody (IgY) against shiga-like toxin (stx) and evaluation of its prophylaxis potency in mice.

OBJECTIVE: Enterohemorrhagic Escherichia coli (E. coli O157: H7) is an enteric pathogen, transmitted through contaminated water and food. Pathogenic factors include bacterial adhesion, invasion of intestinal epithelial and epithelium cells. The pathogenicity of EHEC is due to the production of Shiga-like toxin (Stx). This toxin binds to the ribosome and inhibits the synthesis of proteins. EHEC causes hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The EHEC treatment with antibiotics leads to resistance. The best way to solve this problem is to use specific antibodies and prophylaxis. Egg yolk antibody (IgY) is a suitable method for prophylaxis. Hence, the aim of this study was to investigate the production of IgY against Stx toxin and its prophylaxis. RESULT: The produced antibodies were confirmed by SDS-PAGE and ELISA. IgY was obtained at a concentration of about 5 mg/ml (30 mg of each egg) and a purity of more than 90%. Toxin and antibody challenge was performed in mice. The obtained IgY was able to neutralize the effect of Stx at 2 mg/mice. CONCLUSION: This challenge showed that an antibody produced with an acceptable percentage was able to neutralize the effect of Stx.

Designing a multi-epitope vaccine for cross-protection against Shigella spp: An immunoinformatics and structural vaccinology study.

Shigellosis is a severe diarrheal disease with high mortality and morbidity rate. Until now, there is no approved vaccine against the disease. Therefore, the present study was planned to design a novel multi-epitope vaccine against Shigella spp., the causative agents of the disease based on the immunoinformatic tools. For this end, firstly seven conserved antigens of the bacteria, including IpaA, IpaB, IpaC, IpaD, OmpC, OmpF and VirG were selected. Then, linear B-cell epitope mapping of these proteins was carried out and top-ranked and shared epitopes were selected based on antigenicity, allergenicity, stability, toxicity and physicochemical properties for further analysis. In next step, B-cell derived T-cell epitopes were determined and appropriate epitopes were selected for incorporation into the final construct. Moreover, the selected epitopes and two mucosal adjuvants including ctxB and LT-IIc were joined using appropriate linkers. The three dimensional structure of the final construct was modeled and evaluated in term of structural quality and presence of conformational B-cell epitopes. Furthermore, binding affinity of the proposed vaccine to MHC I and II molecules were evaluated through molecular docking method using Hex 8.0. as well as the stability of the vaccine-MHC complexes was monitored by molecular dynamics method using the NAMD graphical user interface embedded in visual molecular dynamics. Finally, to evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. The results indicated that the proposed vaccine has high structural quality and binding affinity to both MHC I and II molecules. Moreover, molecular dynamics studies confirmed that the vaccine-MHC docked complexes were stable during simulation time. Animal study showed that the proposed protein is able to evoke mice's humoral immune response. In sum, the results suggested that the proposed candidate vaccine could be considered as a promising anti-shigellosis vaccine.

Entrapment of HETS recombinant protein onto PLGA and alginate NPs improves the immunogenicity of the protein against E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) are known as the gastrointestinal pathogens and major causes of enterohemorrhagic colitis since decades ago. There is no efficient approved vaccine against EHEC O157 and non-O157. In the present study, a recombinant candidate vaccine against enterohemorrhagic E. coli (EHEC) O157:H7 entrapped in the sodium alginate and PLGA nanoparticles and the efficiency of the immunization of these formulations were investigated. nanoparticles due to their properties like controlled cargoes release, adjuvanticity, cargo protection, increased bioavailability, etc have been noticed for drug delivery. A chimeric protein composed of HcpA, EspA, Tir and Stx2B antigens was designed, recombinantly expressed, purified and entrapped in nanoparticles. BALB/c mice were administrated with nano-formulated and free proteins. IgG titer, EHEC fecal shedding and the ability of the immune sera to neutralize Stx toxin and inhibit the bacterial attachment to Caco-2 cells were analyzed. Fecal shedding analysis demonstrated that the colonization of the bacteria in the intestine of the mice was reduced significantly (P > 0.01). Immune mice were able to tolerate up to 200 LD50 of the active Stx toxin. About 80% of the bacterial binding capacity to Caco-2 cells was declined, especially in groups immunized with nano-formulations. Considering the importance of EHEC, especially O157 serotype, on public health and the other hand, the lack of an efficient vaccine in this regard, delivery of HETS candidate vaccine with NPs can be applied to prevent the infection by the pathogen.

Immunogenicity evaluation of rBoNT/E nanovaccine after mucosal administration.

OBJECTIVES: The Botulism syndrome is caused by types A to G of botulinum neurotoxins. The binding domains of these neurotoxins are immunogenic and considered as appropriate candidate vaccines. Due to the low immunogenicity of recombinant vaccines, there have been many studies on the use of biocompatible carriers such as chitosan nanoparticles for the delivery of these vaccines. The aim of this study was evaluating the efficiency of chitosan nanoparticles as carriers for a candidate vaccine, binding domain of BoNT/E, through oral and intranasal routes. MATERIALS AND METHODS: Chitosan nanoparticles containing rBoNT/E binding domain, were synthesized via ionic gelation. After administration of the nanoparticles to mice through oral and intranasal routes, antibody titers were assessed by ELISA and, finally, all groups were challenged by active botulinum neurotoxin type E. RESULTS: The groups that received nanoparticles containing the antigen, through oral and intranasal routes, and the group that received the bare antigen orally, were able to tolerate 5×102 folds of MLD. The intranasally immunized mice by the bare antigen were able to tolerate 2×103 folds of the toxin's MLD. CONCLUSION: It seems that the use of chitosan nanoparticles has no significant effect on the protective immunization of the mice against botulinum BoNT/E in either route (P>0.05), even intranasal administration of the bare antigen gives better mice immunization against the toxin.

Gentamicin-Loaded Chitosan Nanoparticles Improve Its Therapeutic Effects on Brucella-Infected J774A.1 Murine Cells.

BACKGROUND: Final elimination of some intracellular bacterial agents, such as Brucella, is often a complex issue and impossible to achieve, primarily due to the presence and survival of the bacteria within phagocytic cells. By penetrating into the cell membrane, drug delivery nanosystems can reduce the number of intracellular bacteria. The aim of this study was to assess the efficacy of chitosan nanoparticles on the delivery of gentamicin into Brucella infected J774A.1 murine cells in vitro. MATERIALS AND METHODS: Chitosan nanoparticles (NPs) were synthesized using ionic gelation technique. The shape, size and charge of NPs, loading rate and release of the drug were investigated. Finally, the effects of gentamicin-loaded chitosan NPs (Gen-Cs) and free gentamicin on J774A.1 murine cells infected with these bacteria were examined. RESULTS: The mean size and charge of NPs were computed as 100 nm and +28mV, respectively. The loading capacity of NPs was 22%. About 70% of the drug was released from NPs during the first 8 hours. Antimicrobial activity of the two formulations showed that MIC (minimum inhibitory concentration) of the Gen-Cs and free drug was 3.1 and 6.25 µg, respectively. The minimum bactericidal concentration of the NPs-loaded drug and free drug was 6.25 and 12.5 µg, respectively. Cell culture analysis revealed that there was a significant reduction in the load of the intercellular bacteria in J774A.1 murine cells in both formulations. CONCLUSION: Our results showed the Gen-Cs have a proper potential for optimal treatment of intracellular bacterial agents.

EspA-loaded mesoporous silica nanoparticles can efficiently protect animal model against enterohaemorrhagic E. coli O157: H7.

In the present study, the application of mesoporous silica nanoparticles (MSNPs) loaded with recombinant EspA protein, an immunogen of enterohaemorrhagic E. coli, was investigated in the case of BALB/c mice immunization against the bacterium. MSNPs of 96.9 ± 15.9 nm in diameter were synthesized using template removing method. The immunization of mice was carried out orally and subcutaneously. Significant immune responses to the antigen were observed for the immunized mice when rEspA-loaded MSNPs were administered in both routes in comparison to that of the antigen formulated using a well-known adjuvant, i.e. Freund's. According to the titretitre of serum IL-4, the most potent humoral responses were observed when the mice were immunized subcutaneously with antigen-loaded MSNPs (244, 36 and 14 ng/dL of IL-4 in the serum of mice immunized subcutaneously or orally by antigen-loaded MSNPs, and subcutaneously by Freund's adjuvant formulated-antigen, respectively). However, the difference in serum IgG and serum IgA was not significant in mice subcutaneously immunized with antigen-loaded MSNPs and mice immunized with Freund's adjuvant formulated-antigen. Finally, the immunized mice were challenged orally by enterohaemorrhagic E. coli cells. The amount of bacterial shedding was significantly reduced in faecesfaeces of the animals immunized by antigen-loaded MSNPs in both subcutaneous and oral routes.