Detachable dissolvable microneedles: intra-epidermal and intradermal diffusion, effect on skin surface, and application in hyperpigmentation treatment.

Delivering bioactive compounds into skin tissue has long been a challenge. Using ex vivo porcine and rat skins, here we demonstrate that a detachable dissolvable microneedle (DDMN) array, a special dissolvable microneedle that allows needle detachment from the base within 2 min post administration, can effectively embed a model compound into epidermis and dermis. Diffusion of the compound from the needle embedding sites to the nearby skin tissue is demonstrated at various post administration periods. The relationship between the time that a conventional dissolvable microneedle array is left on skin without needle detachment from the base and the degree of skin surface abrasion at each microneedle penetration spot is also demonstrated on skin of human volunteers. Co-loading glutathione with vitamin C (vitC) can stabilize vitC in the DDMN. DDMN loaded with vitC and glutathione can help erasing post-acne-hyperpigmentation spots.

Solid Composite Material for Delivering Viable Cells into Skin Tissues via Detachable Dissolvable Microneedles.

Delivering cells to desired locations in the body is needed for disease treatments, tissue repairs, and various scientific investigations such as animal models for drug development. Here, we report the solid composite material that when embedded with viable cells, can temporarily keep cells alive. Using the material, we also show the fabrication of detachable dissolvable microneedles (DMNs) that can instantly deliver viable cells into skin tissue. B16-F10-murine-melanoma (B16-F10) and human-embryonic-kidney-293T (HEK293T) cells embedded in the solid matrix of the hyaluronic/polyvinylpyrolidone/maltose (HA/PVP/maltose) mixture show 50.6 ± 12.0 and 71.0 ± 5.96% survivals, respectively, when kept at 4 °C for 24 h. Detachable DMNs made of the HA/PVP/maltose mixture and loaded with B16-F10-cells were constructed, and the obtained DMN patches could detach the cell-loaded needles into the skin within 1 min of patch application. In vivo intradermal tumorgrafting mice with the DMNs containing 800 cells of B16-F10 developed tumors 10 times bigger in volume than tumors induced by hypodermic needle injection of suspension containing 100,000 cells. We anticipate this work to be a starting point for viable cell encapsulation in the solid matrix and viable cell delivery via DMNs.

Oxidized carbon nanoparticles as an effective protein antigen delivery system targeting the cell-mediated immune response.

Background: The demand for an effective vaccine delivery system that drives a suitable immune response is increasing. The oxidized carbon nanosphere (OCN), a negatively charged carbon nanoparticle, has the potential to fulfill this requirement because it can efficiently deliver macromolecules into cells and allows endosomal leakage. However, fundamental insights into how OCNs are taken up by antigen-presenting cells, and the intracellular behavior of delivered molecules is lacking. Furthermore, how immune responses are stimulated by OCN-mediated delivery has not been investigated. Purpose: In this study, the model protein antigen ovalbumin (OVA) was used to investigate the uptake mechanism and intracellular fate of OCN-mediated delivery of protein in macrophages. Moreover, the immune response triggered by OVA delivered by OCNs was characterized. Methods: Bone-marrow-derived macrophages (BMDMs) from mice were used to study antigen uptake and intracellular trafficking. Mice were immunized using OCN-OVA combined with known adjuvants, and the specific immune response was measured. Results: OCNs showed no cytotoxicity against BMDMs. OCN-mediated delivery of OVA into BMDMs was partially temperature independent process. Using specific inhibitors, it was revealed that intracellular delivery of OCN-OVA does not rely on phagocytosis or the clathrin- and lipid raft/caveolae-mediated pathways. Delivered OVA was found to colocalize with compartments containing MHC class I, but not with early endosomes, lysosomes, and autophagosomes. Immunization of OVA using OCNs in combination with the known adjuvant monophosphoryl lipid A specifically enhanced interferon gamma (IFNγ)- and granzyme B-producing cytotoxic T cells (CTLs). Conclusion: OCNs effectively delivered protein antigens into macrophages that localized with compartments containing MHC class I partially by the temperature independent, but not clathrin- and lipid raft/caveolae-mediated pathways. Increased CD8+ T-cell activity was induced by OCN-delivered antigens, suggesting antigen processing toward antigen presentation for CTLs. Taken together, OCNs are a potential protein antigen delivery system that stimulates the cell-mediated immune response.

Oxidized Carbon Nanosphere-Based Subunit Vaccine Delivery System Elicited Robust Th1 and Cytotoxic T Cell Responses.

Subunit vaccines are safer and more stable than live vaccines although they have the disadvantage of eliciting poor immune response. To develop a subunit vaccine, an effective delivery system targeting the key elements of the protective immune response is a prerequisite. In this study, oxidized carbon nanospheres (OCNs) were used as a subunit vaccine delivery system and tuberculosis (TB) was chosen as a model disease. TB is among the deadliest infectious diseases worldwide and an effective vaccine is urgently needed. The ability of OCNs to deliver recombinant Mycobacterium tuberculosis (Mtb) proteins, Ag85B and HspX, into bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) was investigated. For immunization, OCNs were mixed with the two TB antigens as well as the adjuvant monophosphoryl lipid A (MPL). The protective efficacy was analyzed in vaccinated mice by aerosol Mtb challenge with a virulent strain of Mtb and the bacterial burdens were measured. The results showed that OCNs are highly effective in delivering Mtb proteins into the cytosol of BMDMs and BMDCs. Upon immunization, this vaccine formula induced robust Th1 immune response characterized by cytokine profiles from restimulated splenocytes and specific antibody titer. More importantly, enhanced cytotoxic CD8⁺ T cell activation was observed. However, it did not reduce the bacteria burden in the lung and spleen from the aerosol Mtb challenge. Taken together, OCNs are highly effective in delivering subunit protein vaccine and induce robust Th1 and CD8⁺ T cell response. This vaccine delivery system is suitable for application in settings where cell-mediated immune response is needed.

Gold Nanorods Stabilized by Biocompatible and Multifunctional Zwitterionic Copolymer for Synergistic Cancer Therapy.

A zwitterionic copolymer between methacryloyloxyethyl phosphorylcholine (MPC) and methacrylic acid (MA), PMAMPC is introduced as a potential versatile polymeric stabilizer for gold nanorods (AuNRs). The MA units in the copolymer serve as built-in feature for multiple functionalization, namely introducing additional thiol groups as active sites for binding with the AuNRs and conjugating with doxorubicin (DOX), an anticancer drug via acid-labile hydrazone linkage. The MPC units, on the other hand, provide biocompatibility and antifouling characteristics. The chemically modified PMAMPC can act as an effective stabilizer for AuNRs yielding PMAMPC-DOX-AuNRs with a fairly uniform size and shape with good colloidal stability. In vitro cytotoxicity suggested that PMAMPC can not only improve the AuNRs biocompatibility, but also decrease DOX toxicity to a certain extent. The PMAMPC-DOX-AuNRs were efficiently internalized inside cancer cells and localized in lysosomes, where DOX was presumably acid-triggered released as monitored by confocal laser scanning microscopic analysis and flow cytometry. Furthermore, the combined photothermal-chemo treatment of cancer cells using PMAMPC-DOX-AuNRs exhibited a higher therapeutic efficacy than either single treatment alone. These results suggested that the PMAMPC-DOX-AuNRs could potentially be applied in pH-triggered drug delivery for synergistic cancer therapy.

Molecular detection of Pythium insidiosum from soil in Thai agricultural areas.

Pythium insidiosum is an aquatic fungus-like organism in the kingdom Stramenopila that causes pythiosis in both humans and animals. Human pythiosis occurs in ocular, localized granulomatous subcutaneous and systemic or vascular forms. Individuals whose occupations involve exposure to aquatic habitats have an elevated risk of contracting pythiosis. Previously, we reported the first successful isolation of Pythium insidiosum from aquatic environmental samples by culture including confirmation using molecular methods. In this study, we show that P. insidiosum inhabitats moist soil environments in agricultural areas. A total of 303 soil samples were collected from 25 irrigation sources in the areas nearby the recorded home addresses of pythiosis patients residing in northern provinces of Thailand. P. insidiosum DNA was identified directly from each soil extract by using a nested PCR assay and subsequent phylogenetic analysis of the ribosomal intragenic spacer region. P. insidiosum DNA could be detected from 16 of the 25 soil sources (64%). Conventional culture methods were also performed, however all samples exhibited negative culture results. We conclude that both irrigation water and soil are the natural reservoirs of P. insidiosum. In endemic areas, the exposure to these environmental reservoirs should be considered a risk factor for hosts susceptible to pythiosis.