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Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Immunization programs against SARS-CoV-2 with commercial intramuscular (IM) vaccines prevent disease but not infections. The continued evolution of variants of concern (VOC) like Delta and Omicron has increased infections even in countries with high vaccination coverage. This is due to commercial vaccines being unable to prevent viral infection in the upper airways and exclusively targeting the spike (S) protein that is subject to continuous evolution facilitating immune escape. Here we report a multi-antigen, intranasal vaccine, NanoSTING-NS that yields sterilizing immunity and leads to the rapid and complete elimination of viral loads in both the lungs and the nostrils upon viral challenge with SARS-CoV-2 VOC. We formulated vaccines with the S and nucleocapsid (N) proteins individually to demonstrate that immune responses against S are sufficient to prevent disease whereas combination immune responses against both proteins prevents viral replication in the nasal compartment. Studies with the highly infectious Omicron VOC showed that even in vaccine-naïve animals, a single dose of NanoSTING-NS significantly reduced transmission. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to preventing transmission and ending the pandemic, and (2) an explanation for why hybrid immunity in humans is superior to vaccine-mediated immunity by current IM vaccines.

Activated macrophages are the primary targets in rheumatoid arthritis (RA) management. So, we report efficacious, dual-functional Methotrexate (MTX) loaded folate-conjugated pH-responsive glycol-chitosan nanoparticles (MFGCN) prepared by nano-precipitation and zero-order cross-linking reaction for targeting inflamed arthritic tissue. Physical characterization by DLS, SEM and TEM indicated a spherical, smooth morphology with a diameter ~ 300 nm. 1H NMR and FTIR indicated folic acid conjugation to GC by zero-order cross-linkers. In vitro release kinetics in PBS showed pH-responsive and sustained release behaviour of MFGCN. Enhanced cellular uptake and cytotoxicity of MFGCN in LPS(+)RAW and activated peritoneal macrophages (Mϕ) were observed when compared to LPS(−)RAW cells. MFGCN-induced mitochondrial membrane potential (MMP) perturbations indicated apoptosis. Oxidative stress was evident by significant increase in ROS and RNS, 4 h post incubation with MFGCN. Negligible hemolysis by FGCN and MFGCN on rat RBC’s indicated biocompatibility. In vivo biodistribution of MFGCN in adjuvant-induced arthritis (AIA) rats indicated RA targetability. Prolonged blood circulation coupled with higher concentrations of 99mTc-MFGCN at the arthritic site was observed post 24 h of injection. The gamma scintigraphic image confirmed accumulation of radiolabelled MFGCN in arthritic paw when compared to the non-inflamed paw, confirming the selective uptake of 99mTc-MFGCN by folate-overexpressing macrophages in the arthritic synovium thereby proving its targeted efficacy and theranostic potential. In AIA rats, MFGCN lowers arthritic signs, improves antioxidant response and decreases pro-inflammatory cytokines, suggesting its potential in targeting activated macrophages of synovium.

Respiratory viral infections, especially Influenza (endemic) or SARS-CoV-2 (pandemic since 2020), cause morbidity and mortality worldwide. Despite remarkable progress in the development and deployment of vaccines, they are clearly impacted by the rapid emergence of viral variants. The development of an off-the-shelf, effective, safe, and low-cost drug for prophylaxis against respiratory viral infections is a major unmet medical need. Here, we developed NanoSTING, a liposomally encapsulated formulation of the endogenous STING agonist, 2’-3’ cGAMP, to function as an immunoantiviral. NanoSTING rapidly activates the body’s innate immune system to facilitate a broad-spectrum antiviral response against SARS-CoV-2 and influenza variants in hamsters and mice. We demonstrate that a single intranasal dose of NanoSTING can: (1) treat infections throughout the respiratory system and minimize clinical symptoms, (2) protect against highly pathogenic strains of SARS-CoV-2 (alpha and delta), (3) provide durable protection against reinfection from the same strains without the need for retreatment, (4) prevent transmission of the highly infectious SARS-CoV-2 Omicron strain, and (5) provide protection against both oseltamivir-sensitive and resistant strains of influenza. Mechanistically, administration of NanoSTING rapidly upregulated interferon-stimulated and antiviral pathways in both the nasal turbinates and lung. Our results support using NanoSTING as a thermostable, immunoantiviral with broad-spectrum antiviral properties making it appealing as a therapeutic for prophylactic or early post-exposure treatment.

The objective of the present research was to develop teriflunomide-loaded nanoliposomes by thin-film hydration technique for the management of rheumatoid arthritis. Quality by design approach was applied statistically for the optimization of the teriflunomide-nanoliposomalformulation. The vesicle size, polydispersity index, zeta potential and entrapment efficiency of the optimized teriflunomide-nanoliposomal formulation was found to be 128.92 ± 5.42 nm, 0.206 ± 0.06, +12.6 ± 1.2 mV and 73.94 ± 4.32%, respectively. The optimized formulation exhibited initial burst release followed by sustained release for 24 h. Hemolytic toxicity assay and syringe ability test results revealed that the nanoliposomal formulation was safe and effective after intravenous administration. Rhodamine B loaded nanoliposomal formulation showed significantly higher cellular uptake compared with free Rhodamine B solution in RAW 264.7 cells. In vivo pharmacokinetic study results revealed that the optimized teriflunomide-nanoliposomal formulation followed sustained release and showed higher therapeutic efficacy of the drug at the inflammatory site compared with teriflunomide-solution. Therefore, it can be concluded that the developed teriflunomide-nanoliposomal formulation could improve therapeutic efficacy in the management of rheumatoid arthritis after intravenous administration.

Lignin nanoparticles synthesis is among recent developments in lignin valorization especially for biomedical applications. In this study, a new technique where complete self-assembling of lignin was ensured by simultaneous solvent displacement and flash pH change was used to optimize particle size of blank lignin nanoparticles (BLNPs) for suitability in cell uptake along with maximized yield. To establish BLNPs as drug carrier, safety studies including hemocompatibility, cytotoxicity and elaborate genotoxicity studies on Drosophila melanogaster as a model organism were done. Finally, irinotecan loaded lignin nanoparticles (DLNPs) were synthesized to establish their drug carrying potential and thorough in vitro characterization was performed. BLNPs with controllable size (⁓152 nm), low polydispersity (0.2), maximized yield (65%), negative surface charge (-22 to -23 mV), spherical shape and smooth surface were obtained with acceptable %hemolysis (2%). In vitro cytotoxicity studies revealed that BLNPs were significantly toxic (74.38 ± 4.74%) in human breast adenocarcinoma (MCF-7), slightly toxic (38.8 ± 4.70%) in human alveolar epithelial adenocarcinoma (A-549) and insignificantly toxic (15.89 ± 2.84%) to human embryonic kidney (HEK-293) cells. BLNPs showed concentration dependent early neuronal defects in Drosophila, but nuclei fragmentation and gut cell damage were absent. Sustained release DLNPs with high drug loading reduced the IC

This paper outlines an in vitro evaluation of 3D printed scaffold of polylactic acid (PLA) blended with hydroxyapatite (HAp) and chitosan (CS) for orthopedic tissue engineering applications. In the first stage, selected composition/proportion of PLA–HAp–CS (based on melt flow-ability, mechanical and thermal properties) was 3D printed with fused deposition modeling (FDM) process. The 3D printed scaffolds were used for process capability analysis to ascertain the industrial usability of PLA–HAp–CS composite scaffold for batch production (especially in assembly applications). Further, in the second stage, an in vitro evaluation was performed to investigate the linkages of fibroblast cells for 3D printed scaffold. The results of study outline the rapid increase in growth of fibroblast cells for FDM-printed scaffolds of PLA–HAp–CS thus ensuring its capability of supporting cell adhesion and cell proliferation. Further, the dimensional variations and Shore D hardness of 3D printed scaffolds are under statistically control, with process capability indices (Cp and Cpk ≥ 1).

The present investigation was aimed at developing Teriflunomide (TEF) and Methotrexate (MTX) loaded hydroxyapatite nanoparticles and increasing tolerability towards combination therapy against rheumatoid arthritis by reducing hepatotoxicity.Drug-loaded HAp-NPs were synthesized by wet-chemical precipitation method and optimized by Box-Behnken experimental design. The developed NPs were subjected to in vitro and in vivo characterization. In-vivo pharmacodynamics and biochemical studies were performed on adjuvant- induced arthritis model treated with different formulations; MTX-TEF-SOL, TEF-HAp-NP, MTX-HAp-NP, TEF-MTX-HAp-NP, FOLITRAX-10 and AUBAGIO.The size of the optimized formulations, TEF-HAp-NP and MTX-HAp-NP, was found to be 224.3 ± 83.80 nm and 268.3 ± 73.86 nm with drug loading 53.11 ± 0.84% and 67.04 ± 1.12% respectively. In vitro release of TEF from TEF-HAp-NP (70.41 ± 1.22%) and MTX from MTX-HAp-NP (82.43 ± 1.31%) up to 24 h revealed sustained release pattern. Results of the arthritic assessment study showed a significant (P 0.05) reduction in ankle diameter (61.30 ± 7.42) and arthritis score (2.35 ± 0.24) with a marked restoration of ankle joint micro-architecture in TEF-MTX-HAp-NP treated group. During Hepatotoxicity studies, liver histopathology revealed that the formulation MTX-TEF-HAp-NP was least hepatotoxic with less hepatocyte swelling and fibrous connective tissue proliferation while Folitrax-10 was found to be most hepatotoxic. Biochemical studies revealed that Folitrax-10 significantly (P 0.05) increased the GOT (313.64 ± 16) and GPT level (334.46 ± 13) while insignificant (P 0.05) change in GOT (263.68 ± 17) and GPT (229.38 ± 10) level was recorded with TEF-MTX-HAp-NP.We report that the subcutaneous delivery of TEF-MTX-HAp-NP was most effective as it successfully reduced the dosage by half for maximizing therapeutic efficacy and minimizing side effects. Graphical Abstract ᅟ.

Raloxifene, an anti-osteoporotic drug, is recently approved for prevention of breast cancer in postmenopausal women and thus the drug may be employed to combat the bony adverse effects of letrozole, another anticancer drug. However, the cytotoxic effect of their combination on human breast cancer (MCF-7) and human embryonic kidney (HEK) cell lines is not known. MCF-7 and HEK cell lines were treated with different graded doses of letrozole, raloxifene and their combination, then incubated for 24–48 h. MTT assay was performed to check the cytotoxicity of the drugs. The study indicates that the combination of letrozole and raloxifene possess additive effect in terms of cytotoxicity of cancer cell lines (MCF-7) and negligible effects in normal cell lines (HEK). Our study indicates that the addition of raloxifene doesn't interfere with anticancer efficacy of letrozole rather the combination acted additively for the treatment of breast cancer.

The study purposes to evaluate nanocrystalline biopolymeric nanoparticles encapsulating methotrexate and dexamethasone with high biocompatibility, enhanced therapeutic efficacy and reduced toxicity.Chitosan nanoparticles were prepared by ionic gelation, and Methotrexate (MTX) and Dexamethasone (DEX) were loaded during the preparation and screened for their in vitro efficacy in HEK and RAW264.7 cells, ex vivo and in vivo efficacy.FTIR confirmed the involvement of phosphoric group of sTPP with amine groups of chitosan and also role of hydrogen bonding involved in the preparation of MTXCHNP and DEXCHNP. Controlled release patterns coupled with diffusion of drug were observed in two different buffers (PBS) at pH 7.4 and pH 5.8. The IC50 for MTXCHNP for HEK was 26.1 μg/ml and 7.7 μg/ml for RAW 264.7 cells. In DEXCHNP, the IC50 was 20.12 μg/ml for HEK and 7.37 μg/ml for RAW264.7 cells. Enhanced uptake of FITC-CHNP by RAW cells indicated internalization of nanoparticles by phagocytosis. The enhanced release of drug at lower pH justified increased cytotoxicity. Negligible ex-vivo hemolysis indicated the higher biocompatibility of the nanoparticles.The nanocrystalline biopolymeric nanoparticles were stable, biocompatible and have potential to be administered through i.p. route with minimal toxicity and high efficacy.

Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells. We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting. Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells. These studies provide novel approaches to combat cisplatin resistance in cervical cancer.

The present work evaluates the synergistic anticancer efficacy of bioactive Hydroxyapatite (HA) nanoparticles (HA NPs) loaded with Bendamustine HCl. Hydroxyapatite is a material with an excellent biological compatibility, a well-known fact which was also supported by the results of the Hemolytic studies and a high IC50 value observed in the MTT assay. HA NPs were prepared by the chemical precipitation method and loaded with the drug via physical adsorption. In-vitro release study was performed, which confirmed the sustained release of the drug from the drug loaded HA NPs. MTT assay, Cell Uptake and FACS studies on JURKAT E6.1 cell line and in-vivo pharmacokinetic studies in Wistar rats revealed that the drug loaded HA NPs could be easily internalized by the cells and release drug in a sustained manner. The drug loaded HA NPs showed cytotoxicity similar to the drug solution at 1/10th of the drug content, which indicates a possible synergism between the activity of the anticancer drug and calcium ions derived from the carrier. An increase in intracellular Ca(2+) ions is reported to induce apoptosis in cells. Tumor regression study in Balb/c mice Ehrlich's ascites model presented a similar synergistic efficacy. The drug solution was able to decrease the tumor volume by half, while the drug loaded HA NPs reduced the tumor size by 6 times.

Purpose of the Research: Chitosan nanoparticles (CHNP) are being used to modulate the generation of reactive oxygen species (ROS), as unwarranted generation of ROS can damage proteins, lipid membranes, and DNA of host cells. CHNP possess exceptional abilities to modulate antioxidants and suppress oxidative stress damage caused by the CHNP themselves in normal cells. Methods and Results: CHNP were prepared by ionic gelation in the size range of ~115 nm, with a polydispersity index of 0.365. Doxorubicin (DOX) was encapsulated in CHNP with entrapment efficiency ~48%. The modulation of free radicals and antioxidative enzymes by DOX-loaded CHNP (DLCHNP) was evaluated. The glutathione s-transferase and glutathione levels induced by DLCHNP were lower in Ehrlich ascites carcinoma cells(EACs) cells (6.60 ± 0.02 nM/min/mg protein and 0.92 ± 0.05 nM/min/mg protein, respectively) compared to void CHNP and DOX per se decreased levels of nitric oxide and superoxide dismutase (0.03 ± 0.001 nMoles and 28.84 ± 0.016 Unit/mg protein), elevated levels of GSSG (11.69 ± 0.004 nM/min/mg protein), marginally reduced levels of GSH reductase (1.87 ± 0.002 Unit/mg protein), reduced levels of GPx (31.35 ± 0.022 Unit/mg protein) and significantly enhanced levels of LPO (1.56 ± 0.01 nMoles/mg protein) indicated cellular damage. As observed in DNA fragmentation assay, void nanoparticles did not show any DNA damage whereas DLCHNP caused significant damage. Enhanced gene expressions of Cyt. C and p21 on EACs cells was observed in DLCHNP-treated cells compared to DOX per se. Conclusion: CHNP were not efficient in generating remarkable oxidative stress, but when coupled with a drug (i.e., DLCHNP) severe damage was caused to the cancer cells compared to the free drug. This indicated the potential of our encapsulated nanoparticles in drug delivery.

Despite tremendous efforts to search novel drugs and treatments, cancer continues to be a major health hazard. Modulation of cellular responses to platinating agents has important clinical implications as they are still heavily prescribed against various cancers. First line chemotherapeutic drug used for treating solid tumors, displays dose limiting nephrotoxicity. Natural products are being sought in order to improve the anticancer efficacy of cisplatin. Pectin, a plant polysaccharide have reported strong anti-oxidant and anti-cancer properties. In our study, Pectin- Cisplatin nanoconjugates in size range of (200 ± 20 nm) were synthesized and assessed for their anti-cancer and renoprotective role. The cytotoxicity of Pec-cis nanoconjugates and cis per se was assessed and co-related with the Glutathione-s-transferase level in both, in-vitro (B-16/F-10 mouse melanoma cell line) and in-vivo models (C57BL6 mice) in time (24 hours, 48 hours) and dose dependent (30 μg/ml and 60 μg/ml) manner. Our findings revealed that pectin-cisplatinnanoconjugates exhibited cytotoxicity similar to the cisplatin solution at 1/10th of the cisplatin content, which indicates a possible synergism between the activity of the cisplatin and pectin. MDA and histological findings were corraborated for altered renal function in tumor bearing mice. Enhanced GST level was reported post pectincisplatin nanoconjugates administration which confirmed that pectin-cisplatin treatment ameliorated both functional and histopathologic damage. This was further verified by lowered MDA levels when compared to cisplatinper se. Therefore, our results confirmed that Pectin-cisplatin nanoconjugates exhibited anti-tumor properties and rendered partial protection against cisplatin induced nephrotoxicity thereby proving the biocompatibility of pectin-cisplatin nanoconjugates for therapeutic purposes.