Microneedle-based two-step transdermal delivery of Langerhans cell-targeting immunoliposomes induces a Th1-biased immune response.

Novel Coronavirus is affecting human's life globally and vaccines are one of the most effective ways to combat the epidemic. Transcutaneous immunization based on microneedle (MN) has attracted much attention because of its painlessness, rapidity, high efficiency and good compliance. In this study, CD11c monoclonal antibody-immunoliposomes (OVA@CD11c-ILP) actively targeting to Langerhans cells (LCs) were successfully prepared and were delivered by the microchannels of skin produced by MN to induce an immune response in vivo. OVA@CD11c-ILP could be targeted to LCs by conjugating CD11c monoclonal antibody to the surface of the ILP. OVA@CD11c-ILP promoted the maturation of dendritic cells (DCs) and the uptake and endocytosis of antigen by LCs. Moreover, OVA@CD11c-ILP immunization can significantly inhibit tumor growth and prolong overall survival. Furthermore, a higher antibody's titer ratio of IgG1/IgG2a indicated that the immune response stimulated by this immunization method was Th1-biased and the liposomes showed Th1-type adjuvant effect. In conclusion, the combination delivery system of immunoliposomes and microneedle can significantly improve the efficiency of antigen presentation and effectively activate cellular immune responses in the body, which is expected to be a promising transdermal immune strategy.

A Sequential Therapeutic Hydrogel With Injectability and Antibacterial Activity for Deep Burn Wounds' Cleaning and Healing.

As a severe clinical challenge, escharotomy and infection are always the core concerns of deep burn injuries. However, a usual dressing without multifunctionality leads to intractable treatment on deep burn wounds. Herein, we fabricated a sequential therapeutic hydrogel to solve this problem. Cross-linked by modified polyvinyl alcohol (PVA-SH/ε-PL) and benzaldehyde-terminated F127 triblock copolymers (PF127-CHO), the hydrogel demonstrated excellent mechanical properties, injectability, tissue adhesiveness, antibacterial activity, biocompatibility, and satisfactory wound cleaning through both in vitro and in vivo assays. Additionally, based on the conception of "sequential therapy," we proposed for the first time to load bromelain and EGF into the same hydrogel in stages for wound cleaning and healing. This work provides a strategy to fabricate a promising wound dressing for the treatment of deep burn wounds with injectability and improved patients' compliance as it simplified the process of treatment due to its "three in one" characteristic (antibacterial activity, wound cleaning, and healing effects); therefore, it has great potential in wound dressing development and clinical application.

Polymerized vorinostat mediated photodynamic therapy using lysosomal spatiotemporal synchronized drug release complex.

A combination of photodynamic therapy (PDT) and histone deacetylase inhibitor (HDACis) could potentiate single-mode anti-tumor activity of HDACis or PDT to inhibit tumor relapse and metastasis. However, poor solubility and heterogeneity in cellular uptake and tissue distribution hamper the dual mode antitumor effect. For a controlled drug release of photosensitizers and HDACis in cytoplasm, photosensitizer pyropheophorbide-a (Pyro) encapsulated in polymer polyethylene glycol-b-poly (asparaginyl-vorinostat) (simplified as Pyro@FPPS) are fabricated to achieve their lysosomal spatiotemporal synchronized release. With HDACis modeling PDT in vitro and in vivo, it seems that polymerized Vorinostat encapsulated photosensitizers significantly inhibited the tumor proliferation and metastasis by spatiotemporal synchronized drugs release, and Pyro@FPPS reported here reveals a promising prospect to exert drugs' synergistic effect in a spatiotemporal synchronized manner and can be an effective strategy to inhibit tumor growth, recurrence and metastasis in clinic.

Lysosome activable polymeric vorinostat encapsulating PD-L1KD for a combination of HDACi and immunotherapy.

PD-1/PD-L1 blocking therapy has become one of the most promising methods in the field of tumor treatment. However, it encounters the challenge of immune escape due to the exhaustion of T cells. Studies have shown that the epigenetic regulation drug histone deacetylase inhibitor (HDACi) may be able to reverse exhausted T cells by changing the epigenetic transcription program. Therefore, the combination of epigenetic therapy and PD-1/PD-L1 blockade therapy is expected to reverse the immune escape, whereas the overriding goal should aim at the spontaneous release and synergy of PD-1/PD-L1 blocking siRNA and HDACi. In this study, we develop PDDS{polyethylene glycol-b-asparaginate(diethylenetriamine-vorinostat), (PEG-b-P[Asp(DET-SAHA)n] PPDS)}encapsulating siRNA-PD-L1to provide micelles siRNA-PD-L1-loaded micelles (siRNA@PPDS). Transmission electron microscope (TEM) images demonstrate that siRNA@PPDS micelles presented spherical morphology with a size of about 120 nm; hydrodynamic data analysis indicates pH sensitivity of siRNA@PPDS micelles. The experiments reveal that siRNA@PPDS micelles could be well uptaken by the tumor cells to silence the expression of PD-L1 protein in a dose-dependent manner; compared with the free SAHA, the SAHA-loaded micelles PPDS show higher cytotoxicity to induce tumor cell apoptosis and block cell cycle in G1 phase on melanoma-bearing mice, siRNA@PPDS has shown outstanding inhibition of tumor growth and pulmonary metastasis. By comprehensively activating the immune system, lysosome activable polymeric vorinostat encapsulating PD-L1KD for the combination therapy of PD-L1-KD and HDACIs can be an effective strategy to reverse the unresponsiveness of immune checkpoint inhibitors and a promising treatment to inhibit tumor growth, recurrence, and metastasis in clinic.

Gelated Vorinostat with inner-lysosome triggered release for tumor-targeting chemotherapy.

Histonedeacetylase inhibitor (HDACi) has great potential in targeted antitumor therapy by inhibiting tumor migration, invasion, and metastasis. As one of the typical HDACis, vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) was approved as a therapeutic agent for cancer therapy, however, challenges remain due to their poor solubility, short half-life and low efficiency in cellular penetration. Considering the disadvantages of usual drug carriers, folate and vorinostat bound BSA nanogel (FVBN)was fabricated to implement higher solubility, stability, cellular uptake, and lipase-responsive release. With good dispersion and stability, FVBN significantly increased the cellular uptake of vorinostat through folate-mediated endocytosis. FVBN exhibited comparable cytotoxicity with free SAHA, and the growth of tumor cells was blocked in G1/G0 phase just like SAHA performed in cell cycle arrest tests. Moreover, FVBN not only effectively inhibited the growth of melanoma but also observably prevented pulmonary metastasis of melanoma. In the experiment against nude mice bearing solid ovarian cancer, FVBN showed excellent antitumor effect without liver damage, demonstrating the superiority of gelated and inner-lysosome triggered release strategies to the free SAHA, and it is promising to expand the scope of application of HDACi in clinical cancer therapy.

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Magnetic And pH Dual-Responsive Nanoparticles For Synergistic Drug-Resistant Breast Cancer Chemo/Photodynamic Therapy.

BACKGROUND: Drug resistance is one of the prime reasons of chemotherapy failure in breast cancer and is also an important factor affecting prognosis. PURPOSE: In this study, we constructed a functional magnetic mesoporous silica-based nanocomposite (MMSN) for breast cancer chemotherapy/photodynamic therapy. METHODS: MMSN was characterized by scanning electron microscopy and transmission electron microscopy to observe the morphology. The size distribution and zeta potential of the MSNs were determined using Malvern Particle Size Analyzer. Anti-tumor activity in vitro was investigated by CCK-8 assay, flow cytometry and transwell experiment, and the anti-tumor activity in vivo was probed into by magnetic targeting, toxicity, and antitumor effects in breast cancer-bearing BABL/c nude mice. RESULTS: The results showed that the release of doxorubicin in the nanocomposites was pH sensitive, and the cumulative release rate reached 80.53% at 60 h under acidic conditions. The nanocomposites had a high cellular uptake ability in MCF-7/ADR cells, and the IC50 value of the nanocomposites on MCF-7/ADR cells was 4.23 µg/mL, much smaller than that of free DOX (363.2 µg/mL). The nanocomposites could effectively reverse resistance and induce apoptosis of MCF-7/ADR cells. The blood biochemistry parameters and H&E staining results showed no serious adverse effects after treatment with the nanocomposites. Prussian blue staining showed that the nanocomposites were able to target tumor tissues in tumor-bearing mice under a magnetic field. The combined chemical/photodynamic therapy significantly inhibited tumor growth in vivo. CONCLUSION: Nanocomposites with magnetic and pH dual-responsive performance has shown a promising platform for enhanced drug-resistant breast cancer treatment.

Folate-mediated and pH-responsive chidamide-bound micelles encapsulating photosensitizers for tumor-targeting photodynamic therapy.

Background: Nonspecific tumor targeting, potential relapse and metastasis of tumor after treatment are the main barriers in clinical photodynamic therapy (PDT) for cancer, hence, inhibiting relapse and metastasis of tumor is significant issues in clinic. Purpose: In this work, chidamide as a histone deacetylases inhibitor (HADCi) was bound onto a pH-responsive block polymer folate polyethylene glycol-b-poly(aspartic acid) (PEG-b-PAsp) grafted folate (FA-PEG-b-PAsp) to obtain the block polymer folate polyethylene glycol-b-poly(asparaginyl-chidamide) (FA-PEG-b-PAsp-chidamide, FPPC) as multimodal tumor-targeting drug-delivery carrier to inhibiting tumor cell proliferation and tumor metastasis in mice. Methods: Model photosensitizer pyropheophorbide-a (Pha) was encapsulated by FPPC in PBS to form the polymer micelles Pha@FPPC [folate polyethylene glycol-b-poly(asparaginyl-chidamide) micelles encapsulating Pha]. Pha@FPPC was characterized by transmission electron microscope and dynamic light scattering; also, antitumor activity in vivo and in vitro were investigated by determination of cellular ROS level, detection of cell apoptosis and cell cycle arrest, PDT antitumor activity in vivo and histological analysis. Results: With favorable and stable sphere morphology under transmission electron microscope (TEM) (~93.0 nm), Pha@FPPC greatly enhanced the cellular uptake due to its folate-mediated effective endocytosis by mouse melanoma B16-F10 cells and the yield of ROS in tumor cells induced by PDT, and mainly caused necrocytosis and blocked cell growth cycle not only in G2 phase but also in G1/G0 phase after PDT. Pha@FPPC exhibited lower dark cytotoxicity in vitro and a better therapeutic index because of its higher dark cytotoxicity/photocytotoxicity ratio. Moreover, Pha@FPPC not only significantly inhibited the growth of implanted tumor and prolonged the survival time of melanoma-bearing mice due to both its folate-mediated tumor-targeting and selectively accumulation at tumor site by EPR (enhanced permeability and retention)effect as micelle nanoparticles but also remarkably prevented pulmonary metastasis of mice melanoma after PDT compared to free Pha, demonstrating its dual antitumor characteristics of PDT and HDACi. Conclusion: As a folate-mediated and acid-activated chidamide-grafted drug-delivery carrier, FPPC may have great potential to inhibit tumor metastasis in clinical photodynamic treatment for cancer because of its effective and multimodal tumor-targeting performance as photosensitizer vehicle.

Immunotoxicity assessment of ordered mesoporous carbon nanoparticles modified with PVP/PEG.

With large surface area and three-dimensional pore structure, mesoporous carbon nanoparticles (MCN) have attracted enormous interests as potential drug carriers. However, MCN immunotoxicity has not been clarified clearly up to now. Herein we reported the effect of MCN with and without PVP or DSPE mPEG2000 (PEG) modification on immune cells including dendritic cells (DCs), T lymphocytes and RAW264.7 macrophages in vitro. Furthermore, blood biochemical tests, alexin C3 assay and histological analysis were used to investigate the toxicity of MCN in vivo. The synthesized MCN with average particle size about 90 nm was naturally insoluble in water. Surface modification with PVP (MCN-PVP) or PEG (MCN-PEG) slightly increased the particle size and Zeta potential, and effectively improved the dispersion of mesoporous carbon. MCN, MCN-PVP and MCN-PEG promoted the differentiation and maturation of the DCs, while the levels of secreted TNF-α and IL-6 were significantly suppressed by MCN-PVP and MCN-PEG. These materials significantly induced apoptosis of T lymphocytes. The histopathologic results showed that there was no significant difference between nanoparticles with or without modification. Importantly, the materials deposition was observed in the lung, which could potentially inhibit lung metastasis. In conclusion, the ordered mesoporous carbon nanoparticles superficially modified by PVP or PEG perform well in immunological biocompatibility, and are likely to be a promising candidate as medicine carrier in pharmaceutics and clinic.

Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models.

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS. Given the gain of toxic function in this dominantly inherited disease, lowering SOD1 mRNA and protein is predicted to provide therapeutic benefit. An early generation antisense oligonucleotide (ASO) targeting SOD1 was identified and tested in a phase I human clinical trial, based on modest protection in animal models of SOD1 ALS. Although the clinical trial provided encouraging safety data, the drug was not advanced because there was progress in designing other, more potent ASOs for CNS application. We have developed next-generation SOD1 ASOs that more potently reduce SOD1 mRNA and protein and extend survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice. We demonstrated that the initial loss of compound muscle action potential in SOD1G93A mice is reversed after a single dose of SOD1 ASO. Furthermore, increases in serum phospho-neurofilament heavy chain levels, a promising biomarker for ALS, are stopped by SOD1 ASO therapy. These results define a highly potent, new SOD1 ASO ready for human clinical trial and suggest that at least some components of muscle response can be reversed by therapy.

Synthesis and activity of quinolinylmethyl P1' alpha-sulfone piperidine hydroxamate inhibitors of TACE.

A series of alpha-sulfone piperidine hydroxamate TACE inhibitors 11a-n bearing a quinolinyl methyl P1' group was prepared, and their activity was compared to analogous alpha- and beta-sulfone piperidine hydroxamates with a butynyloxy P1' group. The quinolinyl methyl P1' group affords increased inhibitory enzyme activity relative to the corresponding butynyloxy P1' analogs in the alpha-sulfone piperidine hydroxamate series, and greater selectivity than the corresponding butynyloxy P1' analogs in the beta-sulfone piperidine hydroxamate series.

Structure-based design of TACE selective inhibitors: manipulations in the S1'-S3' pocket.

A series of beta-sulfonyl hydroxamate TACE inhibitors, bearing a butynylamino or a butynyloxy P1' group, was designed and synthesized. Of the compounds investigated, 22 has excellent potency against isolated TACE enzyme, shows good selectivity over MMP-2 and MMP-13, and oral activity in an in vivo mouse model of TNF-alpha production.

Design and synthesis of 3,3-piperidine hydroxamate analogs as selective TACE inhibitors.

Structure-based methods were used to design beta-sulfone 3,3-piperidine hydroxamates as TACE inhibitors with the aim of improving selectivity for TACE versus MMP-13. Several compounds in this series were synthesized and evaluated in enzymatic and cell-based assays. These analogs exhibit excellent in vitro potency against isolated TACE enzyme and show good selectivity for TACE over the related metalloproteases MMP-2, -13, and -14.

Identification of potent and selective TACE inhibitors via the S1 pocket.

By focusing on the P1 portion of the piperidine beta-sulfone ligands we identified a motif that induces selectivity and resulted in a series of TACE inhibitors that demonstrated excellent in vitro potency against isolated TACE enzyme and excellent selectivity over MMPs 1, 2, 9, 13, and 14.

Design and synthesis of butynyloxyphenyl beta-sulfone piperidine hydroxamates as TACE inhibitors.

A series of butynyloxyphenyl beta-sulfone piperidine hydroxamate TACE inhibitors was designed and synthesized. The resulting structure-activity relationship and MMP selectivity of the series were examined. Of the compounds investigated, 17s has excellent in vitro potency against isolated TACE enzyme, shows good selectivity over MMP-1, -2, -7, -8, -9, -13, and -14, and oral activity in an in vivo mouse model of TNF-alpha production.

Characterization of (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide, a potent and selective inhibitor of TNF-alpha converting enzyme.

TNF-alpha converting enzyme (TACE) is a validated therapeutic target for the development of oral tumor necrosis factor-alpha (TNF-alpha) inhibitors. Here we report the pre-clinical results and characterization of a selective and potent TACE inhibitor, (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide (TMI-2), in various in vitro and in vivo assays. TMI-2 is a potent TACE inhibitor in an enzymatic FRET assay (IC50=2 nM). It is more than 250-fold selective over MMP-1, -7, -9, -14, and ADAM-10 in vitro. In cell-based assays and human whole blood, TMI-2 inhibits lipopolysaccharide (LPS)-induced TNF secretion with IC50s<1 uM. Importantly, TMI-2 inhibits the spontaneous release of TNF-alpha in human synovium tissue explants of rheumatoid arthritis patients with an IC50 of 0.8 microM. In vivo, TMI-2 potently inhibits LPS-induced TNF-alpha production in mice (ED50=3 mg/kg). In the adjuvant-induced arthritis (AIA) model in rats, treatment with TMI-2 at 30 mg/kg and 100 mg/kg p.o. b.i.d. was highly effective in reducing joint arthritis scores. In a semi-therapeutic collagen-induced arthritis (CIA) model in mice, TMI-2 is highly effective in reducing disease severity scores after oral treatment at 100 mg/kg twice per day. In summary, TMI-2 is a potent and selective TACE inhibitor that inhibits TNF-alpha production and reduces the arthritis scores in pre-clinical models. TMI-2 represents a novel class of TACE inhibitors that may be effective and beneficial in the treatment of rheumatoid arthritis as well as other TNF-mediated inflammatory autoimmune diseases.

Cytosolic phospholipase A2alpha-deficient mice are resistant to collagen-induced arthritis.

Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2alpha (cPLA2alpha) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2alpha plays a key role in the development of CIA, we backcrossed cPLA2alpha-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2alpha-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2alpha-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2alpha-deficient mice. Although the disease scores differed significantly between cPLA2alpha mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2alpha in the pathogenesis of CIA.

CD8 T cells are sufficient to mediate allorecognition and allograft rejection.

Different T cell subsets may play different roles in allorecognition and allograft rejection. It has been suggested that CD8 T cells can only initiate rejection with help from CD4 T cells. Since CD8 T cells may have different requirements for allorecognition and for costimulation, it is important to clarify the role of CD8 cells in rejection. We examined the role of CD8 cells in allorecognition using a TCR transgenic mouse transplantation model. In our study, CD8 cells were able to recognize alloantigens and reject allografts in the absence of help from CD4 T cells. Furthermore our study provides a model to study the mechanisms of CD8-mediated allograft rejection. It may be important in the future, to consider that CD8 T cells may need to be targeted independently of CD4 T cells in strategies used to prevent rejection and induce tolerance.