Double-layered PLGA/HA microneedle systems as a long-acting formulation of polyphenols for effective and long-term management of atopic dermatitis.

Atopic dermatitis (AD) is a chronic, relapsing inflammatory disorder that requires long-term treatment to achieve optimal control. Topical corticosteroids or calcineurin inhibitors are the mainstay of treatment, but the safety and efficacy of their daily use remain a concern. Here, we report a double-layered poly(lactic-co-glycolic acid) (PLGA)/sodium hyaluronate (HA) microneedle (MN) patch as a long-acting formulation for sustained delivery of natural polyphenols, curcumin (CUR) and gallic acid (GA), into the inflamed skin. Upon insertion into the skin, the HA layer is rapidly dissolved within 5 min for triggering GA release; the PLGA tip is embedded into the dermis for sustained release of CUR for 2 months. Initially, CUR and GA are simultaneously released from the MNs to exert synergistic antioxidant and anti-inflammatory effects, thus promptly relieving AD symptoms. After the complete release of GA, the extended CUR release can maintain the improvement obtained for at least 56 days. Our results revealed that compared with the CUR-only MN and untreated AD groups, the administration of CUR/GA-loaded MNs not only rapidly reduced the dermatitis score from Day 2 but also significantly inhibited epidermal hyperplasia and mast cell accumulation, reduced serum IgE and histamine levels, and downregulated reactive oxygen species production in the skin lesions of Nc/Nga mice on Day 56. These findings demonstrated that the double-layered PLGA/HA MN patch can serve as an effective dual-polyphenol delivery system for rapid and long-term management of AD.

Polyphenol-assisted assembly of Au-deposited polylactic acid microneedles for SERS sensing and antibacterial photodynamic therapy.

3D SERS microneedles with self-assembled AuNPs were fabricated with tannic acid (chemical glue and reductant) on polylactic acid microneedles for in-depth chemical and biomolecular analysis, with LOD values below 200 ppb for small molecules and 102 CFU cm-2 for bacteria. The MB/Au-microneedles were used for photodynamic therapy with SERS-monitored photosensitizer degradation.

A Facile Approach for Rapid Prototyping of Microneedle Molds, Microwells and Micro-Through-Holes in Various Substrate Materials Using CO2 Laser Drilling.

CO2 laser manufacturing has served as an enabling and reliable tool for rapid and cost-effective microfabrication over the past few decades. While a wide range of industrial and biological applications have been studied, the choice of materials fabricated across various laser parameters and systems is often confounded by their complex combinations. We herein presented a unified procedure performed using percussion CO2 laser drilling with a range of laser parameters, substrate materials and various generated microstructures, enabling a variety of downstream tissue/cellular-based applications. Emphasis is placed on delineating the laser drilling effect on different biocompatible materials and proof-of-concept utilities. First, a polydimethylsiloxane (PDMS) microneedle (MN) array mold is fabricated to generate dissolvable polyvinylpyrrolidone/polyvinyl alcohol (PVP/PVA) MNs for transdermal drug delivery. Second, polystyrene (PS) microwells are optimized in a compact array for the formation of size-controlled multicellular tumor spheroids (MCTSs). Third, coverglass is perforated to form a microaperture that can be used to trap/position cells/spheroids. Fourth, the creation of through-holes in PS is validated as an accessible method to create channels that facilitate medium exchange in hanging drop arrays and as a conducive tool for the growth and drug screenings of MCTSs.

Transdermal Delivery of Luteinizing Hormone-releasing Hormone with Chitosan Microneedles: A Promising Tool for Androgen Deprivation Therapy.

Long-term administration of luteinizing hormone-releasing hormone analogs (LHRHa) is the main type of androgen-deprivation therapy (ADT) for lethal prostate cancer. A fully insertable microneedle system, composed of embeddable chitosan microneedles and a dissolvable polyvinyl alcohol/polyvinyl pyrrolidone supporting array, was developed for sustained delivery of LHRHa to the skin. A porcine cadaver skin test showed that chitosan microneedles can be fully embedded within the skin and microneedle-created micropores reseal within 7 days. The measured LHRHa loading amount was 73.3±2.8 µg per microneedle patch. After applying goserelin-containing microneedles to mice, serum LH levels increased initially and then declined below baseline at day 7. In contrast, serum testosterone levels increased to reach a peak at day 14 and then declined to a castration level at day 21. Additionally, such a castration level was maintained for 2 weeks. Therefore, transdermal delivery of goserelin with embeddable chitosan microneedles can produce a castrated state in mice. Such a system is a promising, feasible means of delivering ADT.

Efficient delivery of nanoparticles to deep skin layers using dissolvable microneedles with an extended-length design.

Skin pretreatment with microneedles (MNs) increases drug permeation through the skin by creating microchannels in the skin. However, because of skin's inherent elasticity and self-healing ability, these microchannels shrink or reseal rapidly, thus limiting the nanoparticle (NP) delivery efficiency. This study reports dissolvable polyvinyl alcohol/polyvinylpyrrolidone (PVA/PVP) MNs with an extended-length design for the efficient transdermal delivery of NPs. In this system, poly(d,l-lactide-co-glycolide) NPs are encapsulated within the pyramidal structure of the MNs. The extended length of the PVA/PVP MN allows it to counteract skin indentation during insertion, thus enabling complete insertion of the pyramidal structure into the skin to deliver the NPs. In contrast to MN pretreatments that require passive diffusion of NPs through the skin, the extended PVA/PVP MNs can directly bring the NPs into the deeper skin layers, and then rapidly dissolve in 3 min to release the payload. An in vivo transdermal delivery study showed that approximately 90% of the loaded NPs were delivered to the viable epidermis and dermis, whereas only <2% of topically applied NPs were detected in the skin after being treated with a commercial 3M™ MN product. The NPs delivered by the extended MN remained at the insertion site for 5 days, enabling a sustained release of active agents to the diseased tissue. The proposed MN system could be a promising tool for the transdermal delivery of NPs to treat deep skin diseases such as bacterial infections and malignant tumors.

Near-Infrared Light-Activatable Microneedle System for Treating Superficial Tumors by Combination of Chemotherapy and Photothermal Therapy.

Because of the aggressive and recurrent nature of cancers, repeated and multimodal treatments are often necessary. Traditional cancer therapies have a risk of serious toxicity and side effects. Hence, it is crucial to develop an alternative treatment modality that is minimally invasive, effectively treats cancers with low toxicity, and can be repeated as required. We developed a light-activatable microneedle (MN) system that can repeatedly and simultaneously provide photothermal therapy and chemotherapy to superficial tumors and exert synergistic anticancer effects. This system consists of embeddable polycaprolactone MNs containing a photosensitive nanomaterial (lanthanum hexaboride) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone supporting array patch. Because of this supporting array, the MNs can be completely inserted into the skin and embedded within the target tissue for locoregional cancer treatment. When exposed to near-infrared light, the embedded MN array uniformly heats the target tissue to induce a large thermal ablation area and then melts at 50 °C to release DOX in a broad area, thus destroying tumors. This light-activated heating and releasing behavior can be precisely controlled and switched on and off on demand for several cycles. We demonstrated that the MN-mediated synergistic therapy completely eradicated 4T1 tumors within 1 week after a single application of the MN and three cycles of laser treatment. No tumor recurrence and no significant body weight loss of mice were observed. Thus, the developed light-activatable MN with a unique embeddable feature offers an effective, user-friendly, and low-toxicity option for patients requiring long-term and multiple cancer treatments.

Poly-γ-glutamic acid microneedles with a supporting structure design as a potential tool for transdermal delivery of insulin.

Incomplete insertion is a common problem associated with polymer microneedles (MNs) that results in a limited drug delivery efficiency and wastage of valuable medication. This paper presents a fully insertable MN system that is composed of poly-γ-glutamic acid (γ-PGA) MNs and polyvinyl alcohol (PVA)/polyvinyl pyrrolidone (PVP) supporting structures. The PVA/PVP supporting structures were designed to provide an extended length for counteracting skin deformation during insertion and mechanical strength for fully inserting the MNs into the skin. When inserted into the skin, both the supporting structures and MNs can be dissolved in the skin within 4min, thus quickly releasing the entire drug load from the MNs. To evaluate the feasibility and reproducibility of using the proposed system for treating diabetes, we administered insulin-loaded MNs to diabetic rats once daily for 2days. The results indicated that the hypoglycemic effect in the rats receiving insulin-loaded MNs was comparable to that observed in rats receiving subcutaneous insulin injections. The relative pharmacological availability and relative bioavailability of the insulin were in the range of 90-97%, indicating that the released insulin retained its pharmacological activity. We observed no significant differences in the plasma insulin concentration profiles between the first and second administrations, confirming the stability and accuracy of using the proposed MN system for insulin delivery. These results indicated that the γ-PGA MNs containing the supporting structure design enable complete and efficient delivery of encapsulated bioactive molecules and have great potential for the relatively rapid and convenient transdermal delivery of protein drugs. STATEMENT OF SIGNIFICANCE: Incomplete insertion of microneedles largely limits drug delivery efficiency and wastage of valuable medication. To address this problem, we developed a fully insertable poly-glutamic acid microneedles with a supporting structure design to ensure complete and efficient delivery of encapsulated drugs. The supporting structures were designed to provide an extended length for counteracting skin compressive deformation during puncture and mechanical strength for fully inserting the microneedles into the skin. When inserted into the skin, both the supporting structures and microneedles can be dissolved in the skin within 4min, thus quickly releasing the entire drug load. This study demonstrated that the proposed microneedle system featuring this unique design allows more convenient and efficient self-administration of drugs into the skin.

Remotely triggered release of small molecules from LaB6@SiO2-loaded polycaprolactone microneedles.

We established near-infrared (NIR)-light-triggered transdermal delivery systems by encapsulating NIR absorbers, silica-coated lanthanum hexaboride (LaB6@SiO2) nanostructures and the cargo molecule to be released in biodegradable polycaprolactone (PCL) microneedles. Acting as a local heat source when exposed to an NIR laser, these nanostructures cause a phase transition of the microneedles, thereby increasing the mobility of the polymer chains and triggering drug release from the microneedles. On IR thermal images, the light-triggered melting behavior of the LaB6@SiO2-loaded microneedles was observed. By adjusting the irradiation time and the laser on/off cycles, the amount of molecules released was controlled accurately. Drug release was switched on and off for at least three cycles, and a consistent dose was delivered in each cycle with high reproducibility. The designed microneedles were remotely triggered by laser irradiation for the controlled release of a chemotherapeutic drug, doxorubicin hydrochloride, in vivo. This system would enable dosages to be adjusted accurately to achieve a desired effect, feature a low off-state drug leakage to minimize basal effects and can increase the flexibility of pharmacotherapy performed to treat various medical conditions.

Areca users in combination with tobacco and alcohol use are associated with younger age of diagnosed esophageal cancer in Taiwanese men.

BACKGROUND: Whether the habitual use of substances (tobacco, alcohol, or areca nut (seed of the Areca palm)) can affect the age of esophageal squamous cell carcinoma (ESCC) presentation has rarely been examined. METHODS: The study subjects were those who were males and the first time to be diagnosed as ESCC (ICD-9 150) and who visited any of three medical centers in Taiwan between 2000 and 2009. A standardized questionnaire was used to collect substance uses and other variables. RESULTS: Mean age (±SD) at presentation of ESCC was 59.2 (±11.3) years in a total of 668 cases. After adjusting for other covariates, alcohol drinkers were 3.58 years younger to have ESCC than non-drinkers (p = 0.002). A similar result was found among areca chewers, who were 6.34 years younger to have ESCC than non-chewers (p<0.0001), but not among cigarette smokers (p = 0.10). When compared to the group using 0-1 substances, subjects using both cigarettes and alcohol were nearly 3 years younger to contract ESCC. Furthermore, those who use areca plus another substance were 7-8 years younger. Subjects using all three substances had the greatest age difference, 9.20 years younger (p<0.0001), compared to the comparison group. CONCLUSION: Our findings suggest that habitually consuming tobacco, alcohol, and areca nut can influence the age-onset of ESCC. Since the development of ESCC is insidious and life-threatening, our observation is worthy to be reconfirmed in the large-scale and long-term follow-up prospective cohort studies to recommend the screening strategy of this disease.

Synthesis of a novel glycoconjugated chitosan and preparation of its derived nanoparticles for targeting HepG2 cells.

In the study, a novel chitosan (CS) derivative conjugated with multiple galactose residues in an antennary fashion (Gal-m-CS) was synthesized. A galactosylated CS (Gal-CS) was also prepared by directly coupling lactobionic acid on CS. Using an iontropic gelation method, CS and the synthesized Gal-CS and Gal-m-CS were used to prepare nanoparticles (CS, Gal-CS, and Gal-m-CS NPs) for targeting hepatoma cells. TEM examinations showed that the morphology of all three types of NPs was spherical in shape. No aggregation or precipitation of NPs in an aqueous environment was observed during storage for all studied groups, as a result of the electrostatic repulsion between the positively charged NPs. Little fluorescence was observed in HepG2 cells after incubation with the FITC-labeled CS NPs. The intensity of fluorescence observed in HepG2 cells incubated with the Gal-m-CS NPs was stronger than that incubated with the Gal-CS NPs. These results indicated that the prepared Gal-m-CS NPs had the highest specific interaction with HepG2 cells among all studied groups, via the ligand-receptor-mediated recognition.

Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle.

Pluronic block copolymers (PBCs) have been shown to reverse multidrug resistance (MDR) by inhibiting the P-glycoprotein (P-gp) pump in cancer cells. One of the problems encountered with the use of PBCs is that the micelles disassociate at low concentrations. The study focused on the stabilization of PBC L121 micelles by the formation of crosslinks within their outer shells. To form crosslinks, the two terminal alcohols on L121 were first chemically converted into aldehydes (L121-CHO) using the Dess-Martin periodinane. Diamine compounds were then used to bridge the converted aldehyde termini on L121-CHO via conjugated Schiff bases. After crosslinking, the morphology of the L121 micelles remained spherical in shape and the mean particle sizes of the micelles before and after crosslinking were comparable (100nm). After exposure of MDR KBv cells to free rhodamine-123 (R123), the accumulation of R123 in cells was limited due to the function of P-gp. In contrast, crosslinking of L121 micelles within their outer shells significantly reduced their critical micelle concentration and greatly enhanced their stability, while maintaining their ability to inhibit P-gp function in resistant cells. The results indicated that the L121 micelles with shell crosslinks may be useful as a drug delivery vehicle for cancer chemotherapy.

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells.

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system for the treatment of liver cancer.

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles (P/NPs) composed of block copolymers synthesized by poly(gamma-glutamic acid) and poly(lactide). For the potential of targeting liver cancer cells, galactosamine was conjugated on the prepared nanoparticles (Gal-P/NPs). In the in vitro studies, it was found that both the P/NPs and the Gal-P/NPs had a similar release profile of paclitaxel. The activity in inhibiting the growth of HepG2 cells by the Gal-P/NPs was comparable to that of a clinically available paclitaxel formulation (Phyxol), while the P/NPs displayed a significantly less activity (p<0.05). The biodistribution and anti-tumor efficacy of the prepared nanoparticles were studied in hepatoma-tumor-bearing nude mice. It was found that the groups injected with Phyxol, the P/NPs or the Gal-P/NPs significantly delayed the tumor growth as compared to the control group injected with PBS (p<0.05). Among all studied groups, the group injected with the Gal-P/NPs appeared to have the most significant efficacy in the reduction of the size of the tumor. This is because a large number of the Gal-P/NPs were observed at the tumor site, and subsequently released their encapsulated paclitaxel to inhibit the growth of the tumor. The aforementioned results indicated that the Gal-P/NPs prepared in the study had a specific interaction with the hepatoma tumor induced in nude mice via ligand-receptor recognition. Therefore, the prepared Gal-P/NPs may be used as a potential drug delivery system for the targeted delivery to liver cancers.

Preparation of nanoparticles composed of poly(gamma-glutamic acid)-poly(lactide) block copolymers and evaluation of their uptake by HepG2 cells.

In the study, poly(gamma-glutamic acid) (gamma-PGA) and poly(lactide) (PLA) were used to synthesize block copolymers via a simple coupling reaction between gamma-PGA and PLA to prepare self-assembled nanoparticles. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles as a targeting moiety. gamma-PGA, a water-soluble, biodegradable, and non-toxic compound, was produced by microbial fermentation (Bacillus licheniformis, ATCC 9945a) and then was hydrolyzed. The hydrolyzed gamma-PGA with a molecular weight of 4 kDa and a polydispersity of 1.3 was used, together with PLA (10 kDa, polydispersity 1.1), to synthesize block copolymers. The prepared nanoparticles had a mean particle size of about 140 nm with a zeta potential of about -20 mV. The results obtained by the TEM and AFM examinations showed that the morphology of the prepared nanoparticles was spherical in shape with a smooth surface. In the stability study, no aggregation or precipitation of nanoparticles was observed during storage for up to 1 month, as a result of the electrostatic repulsion between the negatively charged nanoparticles. With increasing the galactosamine content conjugated on the rhodamine-123-containing nanoparticles, the intensity of fluorescence observed in HepG2 cells increased significantly. Additionally, the intensity of fluorescence observed in HepG2 cells incubated with the nanoparticles with or without galactosamine conjugated increased approximately linearly with increasing the duration of incubation. In contrast, there was no fluorescence observed in Hs68 cells (without ASGP receptors) incubated with the nanoparticles with galactosamine conjugated. The aforementioned results indicated that the galactosylated nanoparticles prepared in the study had a specific interaction with HepG2 cells via ligand-receptor recognition.