Determination of vat-photopolymerization parameters for microneedles fabrication and characterization of HPMC/PVP K90 dissolving microneedles utilizing 3D-printed mold.

Three-dimensional (3D) printing serves as an alternative method for fabricating microneedle (MN) patches with a high object resolution. In this investigation, four distinct needle shapes: pyramid mounted over a long cube (shape A), cone mounted over a cylinder (shape B), pyramidal shape (shape C), and conical shape (shape D) were designed using computer-aided design (CAD) software with compensated bases of 350, 450 and 550 µm. Polylactic acid (PLA) biophotopolymer resin from eSun and stereolithography (SLA) 3D printer from Anycubic technology were used to print MN patches. The 3D-printed MN patches were employed to construct MN molds, and those molds were used to produce hydroxypropyl methylcellulose (HPMC) and polyvinyl pyrrolidone (PVP) K90 dissolving microneedles (DMNs). Various printing parameters, such as curing time, printing angle, and anti-aliasing (AA), were varied to evaluate suitable printing conditions for each shape. Furthermore, physical appearance, mechanical property, and skin insertion ability of HPMC/PVP K90 DMNs were examined. The results showed that for shape A and C, the suitable curing time and printing angle were 1.5 s and 30° while for shapes B and D, they were 2.0 s and 45°, respectively. All four shapes required AA to eliminate their stair-stepped edges. Additionally, it was demonstrated that all twelve designs of 3D-printed MN patches could be employed for fabricating MN molds. HPMC/PVP K90 DMNs with the needles of shape A and B exhibited better physicochemical properties compared to those of shape C and D. Particularly, both sample 9 and 10 displayed sharp needle without bent tips, coupled with minimal height reduction (< 10%) and a high percentage of blue dots (approximately 100%). As a result, 3D printing can be utilized to custom construct 3D-printed MN patches for producing MN molds, and HPMC/PVP K90 DMNs manufactured by those molds showed excellent physicochemical properties.

Itaconic Acid Cross-Linked Biomolecule Immobilization Approach on Amine-Functionalized Silica Nanoparticles for Highly Sensitive Enzyme-Linked Immunosorbent Assay (ELISA).

Biomolecule immobilization on nanomaterials is attractive for biosensors since it enables the capture of a higher concentration of bioreceptor units while also serving as a transduction element. The technique could enhance the accuracy, specificity, and sensitivity of the analytical measurements of biomolecules. However, it was found that the limitation in chemically binding biomolecules on nanoparticle surfaces could only cross-link between the C-terminal and N-terminal. Here, we report the facile one-step synthesis of amine-functionalized silica nanoparticles (AFSNPs). (3-Aminopropyl)triethoxysilane was used as a precursor to modify the functional surface of nanoparticles via the Stöber process. The biomolecules were immobilized to the AFSNPs through itaconic acid, a novel cross-linker that binds between the N-terminal and N-terminal and potentially improves proteins and nucleic acid immobilization onto the nanoparticle surface. The newly developed immobilization approach on AFSNPs for biomolecular detection enhanced the efficiency of ELISA, resulting in increased sensitivity. It might also be easily used to identify different pathogens for clinical diagnostics.

HPMC/PVP K90 Dissolving Microneedles Fabricated from 3D-Printed Master Molds: Impact on Microneedle Morphology, Mechanical Strength, and Topical Dissolving Property.

Three-dimensional (3D) printing can be used to fabricate custom microneedle (MN) patches instead of the conventional method. In this work, 3D-printed MN patches were utilized to fabricate a MN mold, and the mold was used to prepare dissolving MNs for topical lidocaine HCl (L) delivery through the skin. Topical creams usually take 1-2 h to induce an anesthetic effect, so the delivery of lidocaine HCl from dissolving MNs can allow for a therapeutic effect to be reached faster than with a topical cream. The dissolving-MN-patch-incorporated lidocaine HCl was constructed from hydroxypropyl methylcellulose (HPMC; H) and polyvinyl pyrrolidone (PVP K90; P) using centrifugation. Additionally, the morphology, mechanical property, skin insertion, dissolving behavior, drug-loading content, drug release of MNs and the chemical interactions among the compositions were also examined. H51P2-L, H501P2-L, and H901P2-L showed an acceptable needle appearance without bent tips or a broken structure, and they had a low % height change (<10%), including a high blue-dot percentage on the skin (>80%). These three formulations exhibited a drug-loading content approaching 100%. Importantly, the composition-dependent dissolving abilities of MNs were revealed. Containing the lowest amount of HPMC in its formulation, H901P2-L showed the fastest dissolving ability, which was related to the high amount of lidocaine HCl released through the skin. Moreover, the results of an FTIR analysis showed no chemical interactions among the two polymers and lidocaine HCl. As a result, HPMC/PVP K90 dissolving microneedles can be used to deliver lidocaine HCl through the skin, resulting in a faster onset of anesthetic action.

Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation.

Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells-particularly osteoblasts-are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti-6Al-4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10-100 µg/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants.

Sequential Injection Amperometric System Coupling with Bioreactor for In-Line Glucose Monitoring in Cell Culture Application.

We proposed a specially designed sequential injection (SI) amperometric system coupling with a bioreactor for in-line glucose monitoring in cell culture. The system is composed of three main parts which are the bioreactor, SI system, and electrochemical detection unit. The bioreactor accommodates six individual cell culture units which can be operated separately under different conditions. The SI system enables automatic in-line sampling and in-line sample dilution, with a specially designed mixing unit; therefore, it has the benefits of fast analysis time and less contamination risk. The use of 3D-printed microfluidic components, a mixing channel, and a flow cell helped to reduce operational time and sample volume. A disposable screen-printed electrode (SPE), modified with glucose oxidase (GOD), carbon nanotube, and gold nanoparticle, was used for detection. The developed system provided a linear range up to 3.8 mM glucose in cell culture media. In order to work with cell culture in higher glucose media, the in-line sample dilution can be applied. The developed SI system was demonstrated with mouse fibroblast (L929) cell culture. The results show that glucose concentration obtained from the SI system is comparable with that obtained from the conventional colorimetric method. This work can be further developed and applied for in vitro cell-based experiments in biomedical research.

Automatic Programmable Bioreactor with pH Monitoring System for Tissue Engineering Application.

Tissue engineering technology has been advanced and applied to various applications in the past few years. The presence of a bioreactor is one key factor to the successful development of advanced tissue engineering products. In this work, we developed a programmable bioreactor with a controlling program that allowed each component to be automatically operated. Moreover, we developed a new pH sensor for non-contact and real-time pH monitoring. We demonstrated that the prototype bioreactor could facilitate automatic cell culture of L929 cells. It showed that the cell viability was greater than 80% and cell proliferation was enhanced compared to that of the control obtained by a conventional cell culture procedure. This result suggests the possibility of a system that could be potentially useful for medical and industrial applications, including cultured meat, drug testing, etc.

Characterization of Hydrophilic Polymers as a Syringe Extrusion 3D Printing Material for Orodispersible Film.

The application of hydrophilic polymers in designing and three-dimensional (3D) printing of pharmaceutical products in various dosage forms has recently been paid much attention. Use of hydrophilic polymers and syringe extrusion 3D printing technology in the fabrication of orodispersible films (ODFs) might hold great potential in rapid drug delivery, personalized medicine, and manufacturing time savings. In this study, the feasibility of 3D-printed ODFs fabrication through a syringe extrusion 3D printing technique and using five different hydrophilic polymers (e.g., hydroxypropyl methylcellulose E15, hydroxypropyl methylcellulose E50, high methoxyl pectin, sodium carboxymethylcellulose, and hydroxyethylcellulose) as film-forming polymers and printing materials has been investigated. Rheology properties and printability of printing gels and physicochemical and mechanical properties of 3D-printed ODFs were evaluated. Amongst the investigated hydrophilic polymers, sodium carboxymethylcellulose at a concentration of 5% w/v (SCMC-5) showed promising results with a good printing resolution and accurate dimensions of the 3D-printed ODFs. In addition, SCMC-5 3D-printed ODFs exhibited the fastest disintegration time within 3 s due to high wettability, roughness and porosity on the surface. However, the results of the mechanical properties study showed that SCMC-5 3D printed ODFs were rigid and brittle, thus requiring special packaging to prevent them from any damage before practical use.

Hydroxypropyl Methylcellulose E15: A Hydrophilic Polymer for Fabrication of Orodispersible Film Using Syringe Extrusion 3D Printer.

Extrusion-based 3D printing technology is a relatively new technique that has a potential for fabricating pharmaceutical products in various dosage forms. It offers many advantages over conventional manufacturing methods, including more accurate drug dosing, which is especially important for the drugs that require exact tailoring (e.g., narrow therapeutic index drugs). In this work, we have successfully fabricated phenytoin-loaded orodispersible films (ODFs) through a syringe extrusion 3D printing technique. Two different grades of hydroxypropyl methylcellulose (HPMC E5 and HPMC E15) were used as the film-forming polymers, and glycerin and propylene glycol were used as plasticizers. The 3D-printed ODFs were physicochemically characterized and evaluated for their mechanical properties and in vitro disintegration time. Then, the optimum printed ODFs showing good mechanical properties and the fastest disintegration time were selected to evaluate their drug content and dissolution profiles. The results showed that phenytoin-loaded E15 ODFs demonstrated superior properties when compared to E5 films. It demonstrated a fast disintegration time in less than 5 s and rapidly dissolved and reached up to 80% of drug release within 10 min. In addition, it also exhibited drug content uniformity within United States Pharmacopeia (USP) acceptable range and exhibited good mechanical properties and flexibility with low puncture strength, low Young's modulus and high elongation, which allows ease of handling and application. Furthermore, the HPMC E15 printing dispersions with suitable concentrations at 10% w/v exhibited a non-Newtonian (shear-thinning) pseudoplastic behavior along with good extrudability characteristics through the extrusion nozzle. Thus, HPMC E15 can be applied as a 3D printing polymer for a syringe extrusion 3D printer.

Knockout of glucosidase II beta subunit inhibits growth and metastatic potential of lung cancer cells by inhibiting receptor tyrosine kinase activities.

Glucosidase II (GluII) plays a major role in regulating post-translation modification of N-linked glycoproteins. We have previously reported that the expression of glucosidase II beta subunit (GluIIß) was significantly increased in lung tumor tissues and its suppression triggers autophagy and/or apoptosis. Here, we investigated the role of GluIIß in cell growth, metastatic potential, and receptor tyrosine kinases (RTKs) signaling activity in lung carcinoma cell lines. CRISPR-CAS9 technology was used to knockout the GluIIß encoding gene (PRKSH) in lung carcinoma cells. GluIIß knockout cells exhibited drastically slower growth rates in comparison to non-target transfected cells, particularly with lower concentrations of fetal bovine serum, indicating impairment of their ability to survive under nutritional deprivation. Cell migration and anchorage-independent growth, the fundamental components of cancer cell metastasis, were significantly decreased in GluIIß knockout cells. Knockout of GluIIß increased the sensitivity of lung cancer cells to cisplatin but reduced their sensitivity to gefitinib. Interestingly, knocking out of GluIIß lowered overall RTK signaling activities to less than half of those in non-target transfected cells, which could represent a novel strategy for blocking multiple RTKs in tumor cells in an effort to improve lung cancer treatment.

Glucosidase II beta subunit (GluIIß) plays a role in autophagy and apoptosis regulation in lung carcinoma cells in a p53-dependent manner.

PURPOSE: Glucosidase II plays a major role in regulating the post-translational modification of N-linked glycoproteins. Previously, we found that the beta subunit of glucosidase II (GluIIß) levels are significantly increased in lung carcinoma tissues, indicating a potential role in lung tumorigenesis. Here, we investigated the role of GluIIß in the regulation of autophagy and apoptosis in lung carcinoma- and immortalized human bronchial epithelial-derived cells. METHODS: A selective glucosidase II inhibitor, bromoconduritol, was used to inhibit GluII enzyme activity and a siRNA-based technology was used to suppress the expression of the GluIIß encoding gene PRKCSH in lung carcinoma cells differing in p53 status. Cell viability was assessed using a MTT assay, cell cycle progression was assessed using flow cytometry, autophagy was assessed using Western blotting and apoptosis was assessed using an annexin V-FITC/PI double labeling method. RESULTS: We found that GluIIß inhibition resulted in the induction of autophagy in all cell lines tested, but apoptosis in only wild-type p53 cells. We also found that GluIIß inhibition dose-dependently decreased activation of the EGFR/RTK and PI3K/AKT signaling pathways. Although the apoptosis inducing effect of GluIIß inhibition appeared to be p53-dependent, we found that a combined treatment with lysosomal inhibitors to block autophagy enhanced the apoptotic effect of GluIIß inhibition in both wild-type p53 and p53-null cells. CONCLUSIONS: Our data indicate that GluIIß inhibition results in autophagy and apoptosis in lung carcinoma-derived cells, supporting the hypothesis that this enzyme may play a role in blocking these two tumor suppressive processes. Since blocking autophagy by lysosomal inhibitors enhanced the apoptosis-inducing effect of bromoconduritol, independent of p53 status, their combined use may hold promise for the treatment of cancer, particularly lung cancer.