Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology.

Nature continually refines its processes for optimal efficiency, especially within biological systems. This article explores the collaborative efforts of researchers worldwide, aiming to mimic nature's efficiency by developing smarter and more effective nanoscale technologies and biomaterials. Recent advancements highlight progress and prospects in leveraging engineered nucleic acids and proteins for specific tasks, drawing inspiration from natural functions. The focus is developing improved methods for characterizing, understanding, and reprogramming these materials to perform user-defined functions, including personalized therapeutics, targeted drug delivery approaches, engineered scaffolds, and reconfigurable nanodevices. Contributions from academia, government agencies, biotech, and medical settings offer diverse perspectives, promising a comprehensive approach to broad nanobiotechnology objectives. Encompassing topics from mRNA vaccine design to programmable protein-based nanocomputing agents, this work provides insightful perspectives on the trajectory of nanobiotechnology toward a future of enhanced biomimicry and technological innovation.

Posterior eye delivery of angiogenesis-inhibiting RNA nanoparticles via subconjunctival injection.

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.

Dose-dependent effect on skin permeation of polar and non-polar compounds.

The study of the relationship between the amount of drug applied to the skin and fraction of drug absorbed can improve our understanding of finite-dose percutaneous absorption in the development of topical products and risk assessment of hazardous chemical exposure. It has been previously shown that an increase in the dose applied to the skin leads to a decrease in the fraction of drug permeated the skin (dose-dependent effect). The objective of this research was to examine the dose-dependent effect using permeants of varying physiochemical properties. The dose-dependent effect was studied using human epidermal membrane under finite dose conditions in Franz diffusion cell with model permeants at doses ranging from 0.1 to 200 µg. The dose-dependent effect was evident with model permeants caffeine, corticosterone, dexamethasone, and estradiol, consistent with the relationship of decreasing fraction of dose permeated the skin at increasing the applied dose. However, no significant dose-dependent effect was observed for the polar model permeants urea, mannitol, tetraethyl ammonium, and ethylene glycol, suggesting different transport mechanisms for these permeants. It was also found that, at relatively high doses, estradiol, dexamethasone, and corticosterone could increase the permeation of polar and lipophilic permeants, which could counter the dose-dependent effect under the conditions studied.

Effect of Receptor Solution in Studies of In Vitro Permeation Test (IVPT).

In Vitro Permeation Test (IVPT) is commonly used to evaluate skin penetration of chemicals and performance of dermatological products. For a permeant with low aqueous solubility, an additive that is expected not to alter the skin barrier can be used in the receptor solution to improve permeant solubility. The objective of this study was to (a) evaluate the effects of these additives in IVPT receptor solution on skin permeability of model permeants and skin electrical resistance and (b) determine the solubility of the permeants in these receptor solutions. Bovine serum albumin (BSA), 2-hydroxypropyl-beta-cyclodextrin (HPCD), ethanol, nonionic surfactant Brij-98, and propylene glycol were the additives, and phosphate buffered saline (PBS) was the control. Steady-state skin permeability coefficients and resistances were determined. The receptor solutions examined in this study did not cause a significant increase in skin permeability or decrease in resistance (less than 40 % changes) except 25 % ethanol. The receptor solution containing 25 % ethanol induced an approximately twofold average increase in skin permeability and reduced skin electrical resistance by approximately threefold. The receptor solution of 2.5 % HPCD provided the highest levels of solubility for the model lipophilic permeants, while 0.2 % Brij-98 and 5 % ethanol showed the lowest solubility enhancement from those in PBS.

A novel inclusion complex of oxybenzone with C-methylresorcin[4]arene deters skin permeation.

Oxybenzone (OXB), a very widely used sunscreen ingredient has the potential to block both UVA and UVB but can penetrate through skin. Studies have revealed its presence in the blood and urine of most humans, which may lead to long-term health effects. As the confined cavities of macrocycles can alter the physical and chemical properties of encapsulated guests, in this study, we investigated the formation of host-guest complexes between C-methylresorcin[4]arene and OXB. Combined experimental (NMR spectroscopy, UV/vis absorption, and fluorescence spectroscopy) and theoretical investigation confirmed the formation of a weak host-guest complex that had a 1 : 1 stoichiometry. Furthermore, skin permeation testing revealed that complexation by C-methylresorcin[4]arene significantly reduced the skin permeation of OXB which can potentially limit the harmful effects of this organic sunscreen.

Permeability of Fresh and Frozen Porcine and Human Gingiva and the Effect of Storage Duration.

The gingiva is the target site for some topical drugs, but the permeability of human gingiva has not been systematically evaluated. Pigs are a common animal model for in vitro membrane transport studies. The objectives of this study were to: (a) determine the permeability coefficients of freshly excised human gingiva using model permeants, (b) compare the permeability coefficients of fresh human gingiva with those of fresh porcine gingiva, (c) evaluate the effect of freezing duration on the permeability of porcine gingiva, and (d) compare the permeability coefficients of fresh and cadaver (frozen) human gingiva. A goal was to examine the feasibility of using porcine gingiva as a surrogate for human gingiva. The potential of using frozen tissues in permeability studies of gingiva was also examined. Fresh and frozen porcine gingiva, fresh human gingiva, and frozen cadaver human gingiva were compared in the transport study with model polar and lipophilic permeants. The fresh porcine and human tissues showed similarities in the "permeability coefficient vs. octanol-water distribution coefficient" relationship. The porcine gingiva had a lower permeability than that of the human, with a moderate correlation between the permeability of the fresh porcine and fresh human tissues. The permeability of the porcine tissues for the model polar permeants increased significantly after the tissues were frozen in storage. Moreover, the frozen human cadaver tissue could not be utilized due to the high and indiscriminating permeability of the tissue for the permeants and large tissue sample-to-sample variabilities.

Chitosan capped-gold nanoparticles as skin penetration enhancer for small molecules: A study in porcine skin.

Skin is considered one of the most convenient sites for drug administration. The present study evaluated the effect of gold nanoparticles stabilized by chitosan (CS-AuNPs) and citrate ions (Ci-AuNPs) on skin permeation of sodium fluorescein (NaFI) and rhodamine b base (RhB) as small model hydrophilic and lipophilic permeants, respectively. CS-AuNPs and Ci-AuNPs were characterized by transmitted electron microscopy (TEM) and dynamic light scattering (DLS). Skin permeation was investigated using porcine skin with diffusion cells and confocal laser scanning microscopy (CLSM). The CS-AuNPs and Ci-AuNPs were spherical-shaped nanosized particles (38.4 ± 0.7 and 32.2 ± 0.7 nm, respectively). The zeta potential of CS-AuNPs was positive (+30.7 ± 1.2 mV) whereas that of Ci-AuNPs was negative (-60.2 ± 0.4 mV). The skin permeation study revealed that CS-AuNPs could enhance the permeation of NaFI with enhancement ratio (ER) of 38.2 ± 7.5, and the effect was superior to that of Ci-AuNPs. CLSM visualization suggested that skin permeation was enhanced by improving the delivery through the transepidermal pathway. However, the permeability of RhB, a lipophilic molecule, was not significantly affected by CS-AuNPs and Ci-AuNPs. Moreover, CS-AuNPs had no cytotoxic toward human skin fibroblast cells. Therefore, CS-AuNPs are a promising skin permeation enhancer of small polar compounds.

Iontophoresis on Porcine and Human Gingiva.

PURPOSE: Iontophoresis is a noninvasive method that enhances drug delivery using an electric field. This method can improve drug delivery to the tissues in the oral cavity. The effects of iontophoresis on gingival drug delivery have not been investigated. The objectives of this study were to (a) determine the flux enhancement of model permeants across porcine and human gingiva during iontophoresis, (b) examine the transport mechanisms of gingival iontophoresis, and (c) evaluate the potential of iontophoretically enhanced delivery for three model drugs lidocaine, ketorolac, and chlorhexidine. METHODS: Passive and iontophoretic fluxes were determined with porcine and human gingiva using a modified Franz diffusion cell and model drugs and permeants. To investigate the transport mechanisms of iontophoresis, the enhancement from the direct-field effect was determined by positively and negatively charged model permeants. The electroosmosis enhancement effect was determined with neutral permeants of different molecular weight. The alteration of the gingival barrier due to electropermeabilization was evaluated using electrical resistance measurements. RESULTS: Significant flux enhancement was observed during gingival iontophoresis. The direct-field effect was the major mechanism governing the iontophoretic transport of the charged permeants. Electroosmosis was from anode to cathode. The effective pore radius of the iontophoretic transport pathways in the porcine gingiva was ~0.68 nm. Irreversible electropermeabilization was observed after 2 and 4 h of iontophoresis under the conditions studied. CONCLUSION: Iontophoresis could enhance drug delivery and reduce transport lag time, showing promise for gingival drug delivery.

Lateral Transport During Membrane Permeation in Diffusion Cell: In Silico Study on Edge Effect and Membrane Blocking.

Membrane transport in diffusion cell studies is not one-dimensional from the donor to the receptor. Lateral diffusion within the membrane into the surrounding clamped region can lead to edge effect. Lateral diffusion can also affect the impact of an object blocking the membrane in a diffusion cell. The effects of lateral transport on permeation across a two-layer membrane in diffusion cells were investigated in this study under edge effect and membrane blocking conditions that could be encountered in previous gingiva and hypothetical skin permeation studies. Model simulations of time-dependent and steady-state transport were performed using COMSOL Multiphysics. The simulations indicated edge effect could increase the steady-state flux across the membrane up to 35% with a relatively thick membrane and small diffusion cell opening (e.g., gingiva study). The edge effect decreased when the relative thickness and permeability of the major barrier (top layer in the two-layer membrane) decreased. When the membrane was partially blocked by an object, lateral diffusion within the membrane could mitigate its impact: e.g., when the object was in the receptor, the impact caused by membrane blocking was reduced more than half. Therefore, membrane lateral transport should be considered under certain circumstances in permeation studies using diffusion cells.

Characterization of Porcine Gingiva for Drug Absorption.

Gingiva or gum is a part of the periodontium that surrounds the tooth. Its main function is to provide an effective barrier to both mechanical trauma and bacterial invasion. Gingiva is the target site for some topical drugs. The most common disease in gingiva is periodontal diseases (gum infections). Understanding the gingiva barrier properties could provide insights into approaches to effective drug delivery for the gingiva. Porcine gingiva was chosen as the model in the present membrane transport study. The permeability coefficients of gingiva were determined using a modified Franz diffusion cell with small diffusional area (0.03 cm2) and 12 model permeants with different physicochemical properties. The influences of edge effect and aqueous boundary layers were not observed in the modified diffusion cell setup for the small pieces of gingiva tissue samples. Lipophilic permeants exhibit higher permeability coefficients than hydrophilic permeants. A correlation was observed between the Log permeability coefficient (Log P) and Log octanol-water distribution coefficient (Log Dow) in the analysis. The permeant molecular weight (MW) was also a factor in the Log P vs. Log Dow relationship. The coefficient of Log Dow in this three-factor relationship (0.42) suggested that the gingiva barrier was less lipophilic than octanol.

Long-Term Antibody Release Polycaprolactone Capsule and the Release Kinetics in Natural and Accelerated Degradation.

Although therapy using monoclonal antibodies (mAbs) has been steadily successful over the last 20 years, the means of delivery of mAbs has not been optimized, especially for long-term delivery. Frequent injections or infusions have been the current standard of care. In this study, we have developed a long-term antibody biodegradable implant using a porous polycaprolactone (PCL) capsule. It released bevacizumab (Bev) slowly for 8 months to date. The Bev release kinetics fit a drug release model with experimental data of the diffusion coefficient and partition coefficient through the polymer capsule. Since screening drug release profiles for the long term (>6 months) is time consuming, an accelerated degradation method was used after validating the characteristics of the PCL capsule in natural and accelerated degradation conditions. The correlation of the time period between natural and accelerated degradation was determined. Overall, the study suggests that mAbs can be released from a porous PCL capsule without an effect of the polymer degradation over a long period (∼6 months) and the long-term release kinetics can be determined by the accelerated degradation within 14 days.

Evaluation of in vitro cornea models for quantifying destructive effects of chemicals.

In vitro models are available as alternatives for the Draize eye irritation test. However, most of the alternative models are not quantitative nor designed to evaluate the effects of chemicals on the corneal barrier such as those encountered in pharmaceutical and cosmetic products. The objective of the present study was to investigate tissue electrical resistance to provide sensitive in vitro testing of tissue alteration caused by chemicals in pharmaceutical and cosmetic formulations as a potential eye irritation testing approach. The experimental protocols for effective tissue resistance measurements were examined using two in vitro eye models: porcine cornea and EpiCorneal. In these models, a test chemical was applied to the cornea or EpiCorneal tissue for 1 min, and tissue resistances/conductances were measured at 1-60 min after the application. The changes in conductance of the tissues after exposure to the chemicals were shown to provide quantitative evaluations to the influence of the chemicals. A correlation was found between the two in vitro models. The results suggest that these models can provide quantitative in vitro assessments of eye-irritating chemicals.

Modification of small dissolution chamber system for long-acting periodontal drug product evaluation.

Conventional dissolution testing methods may not be suitable for long-acting periodontal drug products due to the small volume, slow fluid flow rate, and environment in the periodontal pocket. The objective of this study was to evaluate a 3D-printed small volume flow-through dissolution chamber system (modified from a previous study) for biorelevant and dose-discriminating testing. Three periodontal drug products with different dosage forms were tested: Atridox, Arestin, and PerioChip. Modifications were made to suit the specific characteristics of these dosage forms. No significant differences were observed between the % drug release profiles in vitro and in vivo except for Atridox. The differences observed with Atridox could be related to the exposing surface area of the drug product. Similar differences were observed from this effect in COMSOL model simulations. Overall, the drugs show reasonable in vitro-in vivo correlations (R2 ≥ 0.91) with linear regression slopes close to unity. For dose discrimination between 75% and full dosing, significant differences were observed in the drug release data at specific time points of the products (p ≤ 0.05). The present results suggest that a small volume dissolution chamber with slow flow rate could potentially provide biologically relevant and dose-discriminating evaluations for periodontal drug products.

Permeability of Buccal Mucosa.

The buccal mucosa provides an alternative route of drug delivery that can be more beneficial compared to other administration routes. Although numerous studies and reviews have been published on buccal drug delivery, an extensive review of the permeability data is not available. Understanding the buccal mucosa barrier could provide insights into the approaches to effective drug delivery and optimization of dosage forms. This paper provides a review on the permeability of the buccal mucosa. The intrinsic permeability coefficients of porcine buccal mucosa were collected. Large variability was observed among the published permeability data. The permeability coefficients were then analyzed using a model involving parallel lipoidal and polar transport pathways. For the lipoidal pathway, a correlation was observed between the permeability coefficients and permeant octanol/water partition coefficients (Kow) and molecular weight (MW) in a subset of the permeability data under specific conditions. The permeability analysis suggested that the buccal permeation barrier was less lipophilic than octanol. For the polar pathway and macromolecules, a correlation was observed between the permeability coefficients and permeant MW. The hindered transport analysis suggested an effective pore radius of 1.5 to 3 nm for the buccal membrane barrier.

Passive and iontophoretic transport of pramipexole dihydrochloride across human skin microchannels created by microneedles in vitro.

Skin microchannels (MCs) created by microneedles (MNs) provide a promising route for enhancing transdermal drug delivery. This study investigated passive and iontophoretic transport of pramipexole dihydrochloride (PXCl) across skin MCs created by polymer MN patches made of 1:2 polymethyl-vinyl-ether-co-maleic acid (PMVEMA) to polyvinyl alcohol (PVA) ratio. Permeation studies were performed in vitro using excised human skin under the conditions of (i) "poke-and-patch" and "poke-and-release" delivery approaches with varying concentration of PXCl in the formulations, (ii) drug-loaded dissolving MN (DMN) and hydrogel-forming MN (HGMN) type patches and (iii) combination of MNs and iontophoresis. The results showed that DMN patch greatly enhanced transdermal delivery of PXCl for both "poke-and-patch" and "poke-and-release" approaches as compared with the conventional delivery method. PXCl flux mainly resulted from the contribution of MC pathway created in skin and increased with increasing drug amounts in the formulations. Compared to DMN patch, HGMN patch provided more linear sustained drug delivery over 72 h. Electromigration was the main mechanism of PXCl iontophoresis through MCs and flux enhancement was found to be larger for HGMN patch than DMN patch. These results demonstrated the potential application of MN patches individually or combined with iontophoresis as an alternative method for PXCl administration.

Effect of Pulsed Direct Current on Iontophoretic Delivery of Pramipexole across Human Epidermal Membrane In Vitro.

PURPOSE: Pulsed direct current (PDC) iontophoresis, by allowing skin depolarization, was suggested to provide more efficient ion transport, but the extent of its enhancement effect was unclear. PDC could also offer electric-customized drug delivery. This study examined the effect of PDC iontophoresis on transdermal delivery of pramipexole dihydrochloride (PXCl). METHODS: Iontophoretic delivery of PXCl across human epidermal membrane from pH 7.0 solution was conducted in vitro using continuous direct current (DC) and 6- and 12-cycle PDC iontophoresis (0.5 mA/cm2 and total applied duration of 6 h). Different parameters of PDC iontophoresis were studied, including current density (0.1, 0.2 and 0.5 mA/cm2) and on-off current dosing pattern (1 h/3 h, 0.5 h/3.5 h, and 0.2 h/3.8 h). RESULTS: Both 6- and 12-cycle PDC iontophoresis protocols provided modulation of the permeation profile but delivered smaller amounts of PXCl (396 and 400 µg/cm2, respectively) as compared with continuous DC iontophoresis (482 µg/cm2) at 24 h after 0.5 mA/cm2 and 180 mA/cm2 × min current dose application. Increasing applied current density from 0.1 to 0.5 mA/cm2 increased the PDC iontophoretic flux of PXCl linearly from 5.3 to 14.6 µg/cm2·h (R2 = 0.887). Varying the current level and duration but at the same applied current dose (36 mA/cm2 × min), the total amount of PXCl delivered by PDC iontophoresis at 24 h was independent of the on-off dosing pattern studied (114-128 µg/cm2). CONCLUSIONS: The results indicate that PDC iontophoresis can benefit transdermal delivery of PXCl in terms of controlling its permeation but does not enhance iontophoretic transport compared to continuous DC iontophoresis under the conditions studied.

Effect of pH on Iontophoretic Transport of Pramipexole Dihydrochloride across Human Epidermal Membrane.

PURPOSE: Drugs with higher molecular charges generally show higher flux enhancement when electromigration is the main mechanism in transdermal iontophoresis. This study evaluated the effect of decreasing the formulation pH to increase the positive charges of pramipexole dihydrochloride (PXCl) on its iontophoretic transport across skin. METHODS: In vitro transdermal iontophoresis of PXCl in buffer solution isotonized with either sodium chloride or mannitol were performed in a pH range of 3.0-7.0. Experiments of iontophoresis under symmetric condition with respect to donor and receiver pH and passive transport of the drugs after pretreatment with iontophoresis were conducted to investigate the transport mechanism involved. RESULTS: Iontophoretic permeation of PXCl was pH-dependent in drug solution isotonized with mannitol. The iontophoretic flux of PXCl with valence z = +2 at pH 3.0 was half of that of PXCl with z = +1 at pH 7.0. The results suggest that the decrease in PXCl delivery at higher valence at pH 3 was mainly due to pH-dependent selectivity of PX ion permeation across the skin and not electroosmosis. CONCLUSIONS: Skin permselectivity is a significant factor for iontophoretic transport of PXCl, and reducing formulation pH to increase the positive charges on PX ions did not enhance PXCl delivery.

Laser-Activated Drug Implant for Controlled Release to the Posterior Segment of the Eye.

To treat chronic posterior eye diseases, frequent intravitreal injections or sustained-release drug implants are the current standard of care. Sustained-release drug implants often involve burst release of the drugs and the dosage from the implants cannot be controlled after implantation, which may lead to local side effects. The present study attempts to develop a dosage-controllable drug delivery implant that consists of a nanoporous biodegradable PLGA capsule and light-activated liposomes. Controllable drug release from the implant was achieved using a pulsed near-infrared (NIR) laser both in vitro and in vivo. The in vitro drug release kinetics from two different initial dose implants, 1000 and 500 µg, was analyzed by fitting zero-order and first-order kinetics, as well as the Korsmeyer-Peppas and Higuchi models. The 1000 and 500 µg implants fit the first-order and zero-order kinetics model, respectively, the best. The multiple drug releases in the vitreous were determined by an in vivo fluorimeter, which was consistent with the in vitro data. The dose released was also clinically relevant. Histology and optical and ultrasound imaging data showed no abnormality in the eyes received implant treatment, suggesting that the drug delivery system was safe to the retina. This on-demand dose-controllable drug delivery system could be potentially used for long-term posterior eye disease treatment to avoid frequent invasive injections.

Evaluation of Heat Effects on Fentanyl Transdermal Delivery Systems Using In Vitro Permeation and In Vitro Release Methods.

Experimental conditions that could impact the evaluation of heat effects on transdermal delivery systems (TDS) using an in vitro permeation test (IVPT) and in vitro release testing (IVRT) were examined. Fentanyl was the model TDS. IVPT was performed using Franz diffusion cell, heating lamp, and human skin with seven heat application regimens. IVRT setup was similar to IVPT, without using skin. Dissolution study was conducted in a modified dissolution chamber. The activation energy of skin permeation for fentanyl was determined using aqueous solution of fentanyl. In IVPT, the increase of temperature from 32 °C to 42 °C resulted in a 2-fold increase in flux for fentanyl TDS, consistent with the activation energy determined. The magnitude of flux increase was affected by the heat exposure onset time and duration: higher flux was observed when heat was applied earlier or following sustained heat application. Heat induced flux increases could not be observed when inadequate sampling time points were used, suggesting the importance of optimizing sampling time points. Drug release from TDS evaluated using IVRT was fast and the skin was the rate-limiting barrier for TDS fentanyl delivery under elevated temperature.

Evaluation of Heat Effects on Transdermal Nicotine Delivery In Vitro and In Silico Using Heat-Enhanced Transport Model Analysis.

A combined experimental and computational model approach was developed to assess heat effects on drug delivery from transdermal delivery systems (TDSs) in vitro and nicotine was the model drug. A Franz diffusion cell system was modified to allow close control of skin temperature when heat was applied from an infrared lamp in vitro. The effects of different heat application regimens on nicotine fluxes from two commercial TDSs across human cadaver skin were determined. Results were interpreted in terms of transport parameters estimated using a computational heat and mass transport model. Steady-state skin surface temperature was obtained rapidly after heat application. Increasing skin surface temperature from 32 to 42°C resulted in an approximately 2-fold increase in average nicotine flux for both TDSs, with maximum flux observed during early heat application. ANOVA statistical analyses of the in vitro permeation data identified TDS differences, further evidenced by the need for a two-layer model to describe one of the TDSs. Activation energies associated with these data suggest similar temperature effects on nicotine transport across the skin despite TDS design differences. Model simulations based on data obtained from continuous heat application were able to predict system response to intermittent heat application, as shown by the agreement between the simulation results and experimental data of nicotine fluxes under four different heat application regimens. The combination of in vitro permeation testing and a computational model provided a parameter-based heat and mass transport approach to evaluate heat effects on nicotine TDS delivery.