Controlled Release of Lysozyme Using Polyvinyl Alcohol-Based Polymeric Nanofibers Generated by Electrospinning.

Polymeric nanofibers generated via electrospinning offer a promising platform for drug delivery systems. This study examines the application of electrospun polyvinyl alcohol (PVA) nanofibers for controlled lysozyme (LZM) delivery. By using various PVA grades, such as the degree of polymerization/hydrolysis, this study investigates their influence on nanofiber morphology and drug-release characteristics. LZM-loaded PVA monolithic nanofibers having 50% drug content exhibit efficient entrapment, wherein rapid dissolution is achieved within 30 min. The initial burst of LZM from the nanofiber was reduced as the LZM content was lowered. The initial dissolution is greatly influenced by the choice of PVA grade used; fully hydrolyzed PVA nanofibers demonstrate controlled release due to the reduced water solubility of PVA. Furthermore, coaxial electrospinning, which creates core-shell nanofibers with polycaprolactone as a controlled release layer, enables sustained LZM release over an extended period. This study confirms a correlation between PVA characteristics and controlled drug release and provides valuable insights into tailoring nanofiber properties for pharmaceutical applications.

Dry Powder Inhalers for Proteins Using Cryo-Milled Electrospun Polyvinyl Alcohol Nanofiber Mats.

To enable the efficient delivery of drugs to the lungs, the drug particle design for most dry powder inhalers (DPIs) involves reducing the aerodynamic particle size to a few microns using methods such as spray-drying or jet-milling. Stresses, including heat and the shear forces generated by the preparation processes, may result in the degradation and denaturation of drugs such as those based on peptides and proteins. Here, we showed that cryo-milled polyvinyl alcohol nanofiber mats loaded with α-chymotrypsin by electrospinning exhibited suitable inhalation properties for use in DPIs, while maintaining enzymatic activity. The cryo-milled nanofiber mats were porous to fine particles, and the particle size and drug stability depended on the freezing and milling times. The median diameter of the milled fiber mats was 12.6 µm, whereas the mass median aerodynamic diameter was 5.9 µm. The milled nanofiber mats were successfully prepared, while retaining the enzymatic activity of α-chymotrypsin; furthermore, the activity of milled fiber mats that had been stored for 6 months was comparable to the activity of those that were freshly prepared. This novel method may be suitable for the DPI preparation of various drugs because it avoids the heating step during the DPI preparation process.

Advanced Continuous Flow Platform for On-Demand Pharmaceutical Manufacturing.

As a demonstration of an alternative to the challenges faced with batch pharmaceutical manufacturing including the large production footprint and lengthy time-scale, we previously reported a refrigerator-sized continuous flow system for the on-demand production of essential medicines. Building on this technology, herein we report a second-generation, reconfigurable and 25 % smaller (by volume) continuous flow pharmaceutical manufacturing platform featuring advances in reaction and purification equipment. Consisting of two compact [0.7 (L)×0.5 (D)×1.3 m (H)] stand-alone units for synthesis and purification/formulation processes, the capabilities of this automated system are demonstrated with the synthesis of nicardipine hydrochloride and the production of concentrated liquid doses of ciprofloxacin hydrochloride, neostigmine methylsulfate and rufinamide that meet US Pharmacopeia standards.

Evaluation of liposomal behavior in the gastrointestinal tract after oral administration using real-time in vivo imaging.

Liposomes are regarded as promising drug carriers for enhancing the pharmacological effects of poorly absorbed drugs, such as peptides, following oral administration. Liposomal surface modifications by mucoadhesive polymers could improve drug absorption through interactions with the mucus layer. The main purpose of this study was to establish a method of monitoring the behavior of liposomes within the body after oral administration, particularly in the gastrointestinal (GI) tract, using a real-time in vivo imaging system (IVIS) to elucidate the behavior of surface-modified liposomes. Indocyanine green (ICG) was used as a near-infrared dye to label chitosan (CS) or glycol CS (GCS)-modified liposomes, and to observe the dynamic behavior of the liposomes in rats by noninvasive IVIS after oral administration. First, we validated IVIS results of the rat abdomens by comparing them to quantitative measurements of ICG fluorescence intensity in tissue homogenates. Nano-sized small unilamellar vesicles were retained longer than micro-sized multilamellar vesicles in the GI tract. Furthermore, surface-modified liposomes showed longer-term retention in the GI tract than unmodified liposomes in fasted rats. Moreover, surface modification by CS or GCS effectively prevented the excretion of liposomes from the GI tract and prolonged retention in fed rats.

Gelation Factors of Pectin for Development of a Powder Form of Gel, Dry Jelly, as a Novel Dosage Form.

Jellies for oral administration are dosage forms that contain water, as stipulated in the Japanese Pharmacopeia, and heat is generally applied to the jellies during the manufacturing process. Therefore, it is difficult to formulate drugs that may be affected adversely by water and/or heat. To solve this problem, we tried to develop a powder form of gel as a novel dosage form (dry jelly: jelly medicine extemporaneously prepared) that is converted to jelly after addition of water at the time of administration. For this purpose, a basic gel formulation consisting of pectin, glucono-δ-lactone, dibasic calcium phosphate hydrate, and sucrose was investigated to evaluate the critical factors affecting gelation phenomena. The gel form was developed by adjusting the amount of each component of the formulation and of water added. Gelation occurred even with hard water containing metal ions (hardness of approximately 304 mg/L), and no changes in gel hardness occurred. The desired gel hardness could be controlled by adjusting the amount of water. The gel hardness changed over time after the addition of water, but this change did not affect the dissolution behavior of drugs formulated in the dry jelly.

In Vitro and In Vivo Characterization of Drug Nanoparticles Prepared Using PureNano™ Continuous Crystallizer to Improve the Bioavailability of Poorly Water Soluble Drugs.

PURPOSE: The aim of this study was to enhance the dissolution and oral absorption of poorly water-soluble active pharmaceutical ingredients (APIs) using nanoparticle suspensions prepared with a PureNano™ continuous crystallizer (PCC). METHOD: Nanoparticle suspensions were prepared with a PCC, which is based on microfluidics reaction technology and solvent-antisolvent crystallization. Phenytoin, bezafibrate, flurbiprofen, and miconazole were used as model APIs. These APIs were dissolved in ethanol and precipitated by the addition of water and polyvinyl alcohol. Batch crystallization (BC) using a beaker was also performed to prepare the suspensions. Both PCC and BC formulations were freeze-dried before being characterized in vitro and in vivo. RESULTS: The particle sizes of the nanoparticle suspensions prepared with the PCC were smaller than those prepared by BC. The dissolution rate of each API in vitro significantly increased after crystallization. Reducing the particle size of either the BC or PCC formulation led to increased API flux across Caco-2 cell monolayers. PCC preparations showed higher plasma concentrations after oral administration, demonstrating the advantages of a fast dissolution rate and increased interaction with the gastrointestinal tract owing to the smaller particle size. CONCLUSIONS: PCC can continuously produce nanoparticle APIs and is an efficient approach for improving their oral bioavailability.

Inhalation Properties and Stability of Nebulized Naked siRNA Solution for Pulmonary Therapy.

The use of naked unmodified small interfering RNA (N-siRNA) without vector has previously been investigated as a pulmonary therapy. However, little is known regarding stabilities and aerodynamic particle sizes of N-siRNA-containing droplets; nebulizers have not yet been optimized for N-siRNA solutions. Thus, in this study, we investigated the feasibility of inhaled N-siRNA solutions for pulmonary therapy using nebulization. Various nebulizers and N-siRNA concentrations were assessed in terms of siRNA integrity after nebulization, and inhalation properties including aerodynamic particle size were examined. In comparison with ultrasonic-, air-jet-, and vibrating-mesh nebulizers, N-siRNA integrity was not affected by nebulization. Thus, in further experiments, performances of N-siRNA aerosols with different nebulizers and N-siRNA concentrations were evaluated and screened using an aerodynamic particle sizer (APS) which employed the time-of-flight principle or a cascade impactor. Mean mass aerodynamic diameters of N-siRNA-containing droplets from vibrating-mesh nebulizers tended to decrease with increasing N-siRNA concentrations, reflecting the influence of N-siRNA solutions on surface tension, as indicated by contact angles. These data indicate the utility of APS instruments for investigating the nebulized characteristics of expensive drugs including siRNAs and may facilitate the development of N-siRNA inhalation formulations.

Control of Drug Diffusion Behavior of Xanthan and Locust Bean Gum Gel by Agar Gel.

Oral gel formulations are known as easy to administer drug products for patients who have problems taking drugs including those with conditions such as dysphagia. In addition, there are numerous commercially available oral gel products, most of which are immediate-release formulation that release their pharmaceutical ingredient content by diffusion. This study is focused on developing oral gel formulations that reduce the dosing frequency and dosage compared to the conventional types. This is with the aim of facilitating the use of gel formulations for producing pharmaceutical agents with different dose regimens, thereby enhancing patient convenience. Here, we used naturally derived high-molecular-weight agar (Ag), xanthan gum (Xa), and locust bean gum (Lo) as gel bases to prepare a variety of gel membranes, and evaluated the diffusion coefficient of the model substances. The result revealed that the Ag content in the Xa-Lo combination gel concentration-dependently increased the diffusion coefficient. Moreover, these findings were applied in an attempt to mask the taste of intensely bitter levofloxacin. The results indicated that the Xa-Lo combination gel exhibited a significantly superior masking effect to that of the Ag gel. This study demonstrates the feasibility of using oral gel formulations to modulate the controlled-release functionality of pharmaceutical agents.

Characterization of a doxorubicin liposome formulation by a novel in vitro release test methodology using column-switching high-performance liquid chromatography.

A novel in vitro release test methodology for a liposome formulation was developed using a column-switching high-performance liquid chromatography (HPLC) system. Doxorubicin (DXR) liposome formulations were used as a model. A DXR liposome formulation was dispersed into a release medium, and the dispersion fluid was directly injected at predetermined time points into the column-switching HPLC system. To evaluate the release profile, this system can be used for determining the released and encapsulated DXR in the liposome formulation separately. Comparison with a conventional in vitro release test methodology by dialysis revealed that the methodology developed by column-switching HPLC had no rate-limiting process of membrane permeation of the drug (which is occasionally observed in the dialysis method). The in vitro release profiles of DXR liposome formulations were well characterized using the method developed by column-switching HPLC, and different in vitro release characteristics were revealed. The developed method did not require a large amount of sample or a complicated pretreatment. In addition, the developed column-switching HPLC system was applicable for characterization of the encapsulation profile of liposome formulations.

Development of Polyvinyl Alcohol (PVA) Nanofibers Containing Cationic Lipid/siRNA Complexes via Electrospinning: The Impact of PVA Characterization.

This study aimed to develop polyvinyl alcohol (PVA) nanofibers encapsulating 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/siRNA complexes via electrospinning for the delivery of nucleic acid-based drugs. It also focused on the influence of the intrinsic properties of PVA on the efficacy of the system. PVA nanofibers, with diameters of 300-400 nm, were obtained, within which the siRNA remained intact and the DOTAP/siRNA complexes were uniformly dispersed. By incorporating DOTAP/siRNA complexes into the PVA nanofibers and assessing the impact of their RNA interference (RNAi) activity in A549-Luc cells, a stable inhibition of luciferase expression was observed. An examination of the nanofiber preparation process revealed that even when DOTAP or siRNA were added separately to the PVA solution without forming complexes, the RNAi effect was retained. The DOTAP/siRNA complexes released from the PVA nanofibers were internalized by the cells, with some PVA residues remaining on their surfaces. The significance of the degree of hydrolysis and polymerization of PVA on the performance of nanofibers was highlighted. Notably, PVA with a low degree of hydrolysis substantially enhanced RNAi effects, with luciferase expression inhibition reaching 91.5 ± 0.7%. Nanofibers made of PVA grades with anionic or cationic modifications were also evaluated, suggesting that they affect the efficacy of siRNA delivery. The insights obtained suggest avenues for future research to optimize drug delivery systems further.

Cryomilled electrospun nanofiber mats containing d-mannitol exhibit suitable for aerosol delivery of proteins.

Dry powder inhalers (DPIs) are the first choice for inhalation drug development. However, some conventional DPI formulation processes require heating, which may damage high molecular weight drugs such as proteins and nucleic acids. In this study, we propose a novel DPI preparation process that avoids the use of heat. Dry powders were prepared by cryomilling nanofiber mats composed of polyvinyl alcohol, D(-)-mannitol (Man), and α-chymotrypsin (α-Chy) as the model drug using the electrospinning method. The addition of Man conferred high dispersibility and excellent in vitro aerosol performance to the nanofiber mat powder in a very short milling time (less than 0.5 min) as assessed using the Andersen cascade impactor. Powders were classified according to the degree of friability, and among these, nanofiber mats containing 15 % Man and milled for 0.25 min exhibited the highest aerosol performance. Nanofiber mats containing Man milled for less than 0.5 min also exhibited greater α-Chy enzymatic activity than a nebulized α-Chy solution. Furthermore, single inhalation induced no significant lung tissue damage as evidenced by lactate dehydrogenase activity assays of mouse bronchoalveolar lavage fluid. This novel DPI formulation process may facilitate the safe and efficient inhalational delivery of therapeutic proteins.

Stable and inhalable powder formulation of mRNA-LNPs using pH-modified spray-freeze drying.

A powder formulation for mucosal administration of mRNA-encapsulated lipid nanoparticles (mRNA-LNPs) is expected to be useful for respiratory diseases. Although freeze-drying is widely used to obtain solid formulations of mRNA-LNPs, highly hydrosoluble cryoprotectants, such as sucrose are necessary. However, sucrose is not a suitable excipient for inhalation powders because of its hygroscopic and deliquescence properties. Spray freeze-drying (SFD) is a method to produce inhalable powder formulation. In this study, we prepared inhalable powder formulations of mRNA-LNPs without deliquescence excipients using pH-modified SFD, which strengthens the interaction between mRNA and ionizable lipids of LNPs by acidic pH modifier, leading to retention of the encapsulated structure of mRNA-LNPs even after SFD. Powdered mRNA-LNPs were suitable for inhalation, and mRNA was encapsulated in LNPs after SFD. The mRNA encapsulation efficiency and mRNA transfection efficiency of pH-modified SFD-mediated powdered mRNA-LNPs were higher than those of conventional SFD, although they were significantly lower than those of liquid intact mRNA-LNPs. However, after long-term storage, the powdered formulation of the mRNA-LNPs exhibited higher mRNA transfection efficiency than liquid mRNA-LNP. Powdered mRNA-LNPs also exerted their function in air-liquid interface cultivation and in vivo intratracheal administration. Collectively, the powder formulation of mRNA-LNPs especially prepared by SFD is expected to be applied for dry powder inhalers.

Endocytosis-like uptake of surface-modified drug nanocarriers into giant unilamellar vesicles.

We had previously developed surface-modified poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for use as a cellular drug delivery system. The cellular uptake of PLGA-NPs was mediated predominantly by endocytosis, and this uptake was increased by surface modifications with polymers, such as chitosan (CS) and polysorbate 80 (P80). In the present study, we prepared a cell-sized giant unilamellar vesicle (GUV) that mimics a cell membrane to investigate the interaction between cell membranes and NPs. Endocytosis-like uptake of NPs into a GUV was observed when the NPs were modified with nonionic surfactant P80 probably due to change in viscoelasticity and enhanced fusion activity of the membrane induced by P80. In contrast, unmodified NPs and those modified with CS were not internalized into a GUV. These results suggest that surface properties of PLGA-NPs are an important formulation parameter for their interaction with lipid membranes.

Nanomedical system for nucleic acid drugs created with the biodegradable nanoparticle platform.

Nanomedical applications of biodegradable poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) developed are discussed in this review. A surface-functionalized PLGA NP platform for drug delivery was established to encapsulate a number of macromolecular drugs such as peptides and nucleic acids as well as low-molecular-weight drugs by the emulsion solvent diffusion method. The interaction of PLGA NPs with cells and tissues could be controlled by changing the surface properties of NPs, suggesting their potential utility for the intracellular drug delivery of nucleic acid-based drugs. Furthermore, orally administered NF-κB decoy oligonucleotide-loaded CS-PLGA NPs are also useful in treating experimental colitis. These approaches using surface-modified PLGA NPs could be able to open new possibilities for nucleic acid-based drug delivery via noninvasive administration method.

Hydroxypropyl-ß-cyclodextrin Enhances Oral Absorption of Silymarin Nanoparticles Prepared Using PureNano™ Continuous Crystallizer.

The oral bioavailability of drugs is limited by factors such as poor membrane permeability, low solubility, and low dissolution rate. Silymarin (SLM) is a health-food active ingredient that is good for immunosuppression and tumor suppression. However, obtaining a good oral bioavailability is difficult owing to its poor solubility and low dissolution ability. To overcome these concerns, we previously prepared SLM nanoparticles (NPs) using the high-pressure crystallization method (PureNanoTM) and freeze-dried them with erythritol (Ery) or hydroxypropyl-ß-CyD (HP-ß-CyD) as a water-soluble dispersion stabilizer. In the present study, we investigated the mechanism underlying the improved absorption of SLM/hypromellose (HPMC)/HP-ß-CyD NPs after oral administration. The SLM/HPMC nano-suspension prepared using PureNanoTM exhibited a narrow size distribution. The size of the SLM/HPMC/HP-ß-CyD NPs was approximately 250 nm after hydration. The SLM/HPMC/HP-ß-CyD NPs were rapidly dissolved, and demonstrated a high solubility under supersaturated conditions. Additionally, they exhibited good wettability and their membrane permeability was improved compared with that of SLM original powder. These results suggest that the formulation of SLM NPs using PureNanoTM and freeze-drying with HP-ß-CyD improves the absorption of SLM after oral administration by enhancing solubility, wettability, and membrane permeability.

Design and evaluation of folate-modified liposomes for pulmonary administration in lung cancer therapy.

Pulmonary drug administration for the treatment of lung cancer is useful because the drug is directly delivered to the lung tissues with minimal invasiveness and higher efficiency compared to other conventional methods. However, it is critical to enhance drug accumulation in the lung cancer tissues to achieve sufficient therapeutic efficacy. The submicron-sized liposome (ssLip) preparation is one of the most promising approaches to enhance drug accumulation in the lungs; however, ssLips prepared for conventional inhalation do not have tumour selectivity. Therefore, in this study, we prepared folate (FA)-modified ssLip (FA-ssLip) to enhance drug accumulation in folate receptor (FR)-expressing lung cancer cells, and evaluated its physicochemical properties and potential as a drug carrier in pulmonary administration. In addition, we prepared rapamycin (RM-an autophagy-inducing anticancer drug)-loaded FA-ssLip (RM/FA-ssLip) and investigated its anti-tumour effect. FA-ssLip showed excellent nanoparticle properties with submicron size (approximately 120 nm) and high lung accumulation in lung cancer mouse model-bearing LL2 cells-a mouse Lewis lung carcinoma cell line. RM/FA-ssLip showed significant cytotoxic activity in FR-expressing cancer cells. In addition, pulmonary administration of RM/FA-ssLip extended the survival of LL2 cell tumour-bearing mice. Taken together, our results suggest the potential of FA-ssLip as a pulmonary drug carrier for the efficient treatment of lung cancer.

Improvements in transfection efficiency with chitosan modified poly(DL-lactide-co-glycolide) nanospheres prepared by the emulsion solvent diffusion method, for gene delivery.

This study sought to evaluate the in vitro transfection efficiency of plasmid DNA (pDNA)-loaded chitosan-modified poly(DL-lactide-co-glycolide) nanospheres (CS-PLGA NS) in a gene-delivery system. Using the emulsion solvent diffusion (ESD) method, pDNA-loaded PLGA NS was prepared and the surface of the PLGA NS was modified by binding to CS. Gene transfection ability of CS-PLGA NS was examined in A549 cells. The luciferase gene was used as a reporter gene. The pattern of luciferase activity by pDNA-loaded CS-PLGA NS was initially weak, but gradually grew stronger before decreasing activity. These phenomena should be in accordance with the sustained-release profile of pDNA from PLGA NS in the cytosol and the pDNA protection against DNase. Positively charged CS-PLGA NS was found, by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, not to exhibit cytotoxicity on A549 cells. These results suggest that CS-PLGA NS are potential contributors to efficient pDNA delivery due to their increased interactions with cells and lack of cytotoxic effects.

Intracellular drug delivery using polysorbate 80-modified poly(D,L-lactide-co-glycolide) nanospheres to glioblastoma cells.

We investigated doxorubicin (DOX)-loaded nanospheres (NS) formulated using a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), for targeted chemotherapy of brain tumours. A nonionic surfactant, polysorbate 80 (P80), was used to modify the surfaces of PLGA NS to improve cellular drug delivery. DOX-loaded PLGA NS were formulated by emulsion solvent diffusion and characterised for DOX encapsulation and in vitro release. The effectiveness of DOX-loaded P80-PLGA NS was investigated in A172 human glioblastoma cells. The drug release pattern was dependent on the pH of the medium. Quantitatively, the cellular uptake of NS was significantly increased by P80 surface modification compared with unmodified NS. Confocal laser scanning microscopy studies revealed that DOX was released from NS following accumulation in the cell nuclei. DOX-loaded P80-PLGA NS could significantly inhibit both DOX efflux from the cells and cell proliferation compared with a DOX solution.

Oral mucus-penetrating PEGylated liposomes to improve drug absorption: Differences in the interaction mechanisms of a mucoadhesive liposome.

We investigated the feasibility of densely polyethylene glycol (PEG2000)-modified liposomes as mucus-penetrating particles (MPPs) for oral delivery of systemically absorbed peptides. The oral absorption of MPPs and mucoadhesive liposomes modified with glycol chitosan (GCS) was compared. In an in vitro artificial mucus model, the densely PEGylated liposomes showed mucus permeability. Intracellular uptake of liposomes was evaluated in a Caco-2 and mucus-secreting Caco-2/HT29 co-culture. Intracellular uptake of MPPs was unaffected by mucus in the co-culture system, whereas the cellular uptake of GCS-liposomes was lower with a mucus layer than in Caco-2 alone. Rat in vivo oral absorption of liposomes was evaluated by using fluorescein isothiocyanate dextran (FD) as a model peptide drug. Oral absorption was higher for densely PEGylated than for unmodified liposomes and was PEG-concentration dependent, but excessive PEGylation decreased FD blood concentration. PEGylated liposomes incorporating spermine (SPM) as an absorption enhancer were then designed and showed the highest in vivo absorption of FD of all tested formulations. The pharmacological effects of the oral liposomes were evaluated by using elcatonin and did not correlate with FD oral absorption. The non-PEGylated SPM liposomes showed the highest pharmacological effect, suggesting the need for drug-specific optimization of liposomal components and surface modifiers.

Monovalent antibody-conjugated lipid-polymer nanohybrids for active targeting to desmoglein 3 of keratinocytes to attenuate psoriasiform inflammation.

To improve the treatment of psoriasiform inflammation, we developed actively targeted nanocarriers loaded with the phosphodiesterase 4 inhibitor AN2728. Methods: Phospholipid-poly(lactic-co-glycolic acid) nanohybrids were prepared and conjugated with monovalent anti-desmoglein 3 antibody to bind keratinocytes. Results: The actively targeted nanohybrids were 229 nm in mean size with a nearly neutral surface charge. Flow cytometry and confocal microscopy showed a 9-fold increase in keratinocyte uptake of targeted nanohybrids relative to non-targeted nanoparticles. The nanoparticles localized mainly in lysosomes after internalization. AN2728-loaded antibody-conjugated nanocarriers inhibited cytokine/chemokine overexpression in activated keratinocytes without affecting cell viability. The targeted nanohybrids also suppressed neutrophil migration by reducing CXCL1 and CXCL2 release from keratinocytes. Following subcutaneous administration in mice, the nanohybrids distributed to the epidermis and hair follicles. In a psoriasis-like skin mouse model, the actively targeted nanoparticles were superior to free drug and non-targeted nanoparticles in mitigating skin inflammation. Intervention with the targeted nanosystem reduced the epidermal thickness of the psoriasiform lesion from 191 to 42 µm, decreased the Psoriasis Area Severity Index by 74%, restored barrier function, and returned chemokine levels to baseline. Conclusions: Our developed nanosystem was safe and demonstrated efficient targeting properties for the treatment of cutaneous inflammation.