A measles and rubella vaccine microneedle patch in The Gambia: a phase 1/2, double-blind, double-dummy, randomised, active-controlled, age de-escalation trial.

BACKGROUND: Microneedle patches (MNPs) have been ranked as the highest global priority innovation for overcoming immunisation barriers in low-income and middle-income countries. This trial aimed to provide the first data on the tolerability, safety, and immunogenicity of a measles and rubella vaccine (MRV)-MNP in children. METHODS: This single-centre, phase 1/2, double-blind, double-dummy, randomised, active-controlled, age de-escalation trial was conducted in The Gambia. To be eligible, all participants had to be healthy according to prespecified criteria, aged 18-40 years for the adult cohort, 15-18 months for toddlers, or 9-10 months for infants, and to be available for visits throughout the follow-up period. The three age cohorts were randomly assigned in a 2:1 ratio (adults) or 1:1 ratio (toddlers and infants) to receive either an MRV-MNP (Micron Biomedical, Atlanta, GA, USA) and a placebo (0·9% sodium chloride) subcutaneous injection, or a placebo-MNP and an MRV subcutaneous injection (MRV-SC; Serum Institute of India, Pune, India). Unmasked staff ransomly assigned the participants using an online application, and they prepared visually identical preparations of the MRV-MNP or placebo-MNP and MRV-SC or placebo-SC, but were not involved in collecting endpoint data. Staff administering the study interventions, participants, parents, and study staff assessing trial endpoints were masked to treatment allocation. The safety population consists of all vaccinated participants, and analysis was conducted according to route of MRV administration, irrespective of subsequent protocol deviations. The immunogenicity population consisted of all vaccinated participants who had a baseline and day 42 visit result available, and who had no protocol deviations considered to substantially affect the immunogenicity endpoints. Solicited local and systemic adverse events were collected for 14 days following vaccination. Unsolicited adverse events were collected to day 180. Age de-escalation between cohorts was based on the review of the safety data to day 14 by an independent data monitoring committee. Serum neutralising antibodies to measles and rubella were measured at baseline, day 42, and day 180. Analysis was descriptive and included safety events, seroprotection and seroconversion rates, and geometric mean antibody concentrations. The trial was registered with the Pan African Clinical Trials Registry PACTR202008836432905, and is complete. FINDINGS: Recruitment took place between May 18, 2021, and May 27, 2022. 45 adults, 120 toddlers, and 120 infants were randomly allocated and vaccinated. There were no safety concerns in the first 14 days following vaccination in either adults or toddlers, and age de-escalation proceeded accordingly. In infants, 93% (52/56; 95% CI 83·0-97·2) seroconverted to measles and 100% (58/58; 93·8-100) seroconverted to rubella following MRV-MNP administration, while 90% (52/58; 79·2-95·2) and 100% (59/59; 93·9-100) seroconverted to measles and rubella respectively, following MRV-SC. Induration at the MRV-MNP application site was the most frequent local reaction occurring in 46 (77%) of 60 toddlers and 39 (65%) of 60 infants. Related unsolicited adverse events, most commonly discolouration at the application site, were reported in 35 (58%) of 60 toddlers and 57 (95%) of 60 infants that had received the MRV-MNP. All local reactions were mild. There were no related severe or serious adverse events. INTERPRETATION: The safety and immunogenicity data support the accelerated development of the MRV-MNP. FUNDING: Bill & Melinda Gates Foundation.

Microneedle patch-based enzyme-linked immunosorbent assay to quantify protein biomarkers of tuberculosis.

There is a clinical need for differential diagnosis of the latent versus active stages of tuberculosis (TB) disease by a simple-to-administer test. Alpha-crystallin (Acr) and early secretory antigenic target-6 (ESAT-6) are protein biomarkers associated with the latent and active stages of TB, respectively, and could be used for differential diagnosis. We therefore developed a microneedle patch (MNP) designed for application to the skin to quantify Acr and ESAT-6 in dermal interstitial fluid by enzyme-linked immunosorbent assay (ELISA). We fabricated mechanically strong microneedles made of polystyrene and coated them with capture antibodies against Acr and ESAT-6. We then optimized assay sensitivity to achieve a limit of detection of 750 pg/ml and 3,020 pg/ml for Acr and ESAT-6, respectively. This study demonstrates the feasibility of an MNP-based ELISA for differential diagnosis of latent TB disease.

An ultra-low-cost electroporator with microneedle electrodes (ePatch) for SARS-CoV-2 vaccination.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other pathogens with pandemic potential requires safe, protective, inexpensive, and easily accessible vaccines that can be developed and manufactured rapidly at a large scale. DNA vaccines can achieve these criteria, but induction of strong immune responses has often required bulky, expensive electroporation devices. Here, we report an ultra-low-cost (<1 USD), handheld (<50 g) electroporation system utilizing a microneedle electrode array ("ePatch") for DNA vaccination against SARS-CoV-2. The low cost and small size are achieved by combining a thumb-operated piezoelectric pulser derived from a common household stove lighter that emits microsecond, bipolar, oscillatory electric pulses and a microneedle electrode array that targets delivery of high electric field strength pulses to the skin's epidermis. Antibody responses against SARS-CoV-2 induced by this electroporation system in mice were strong and enabled at least 10-fold dose sparing compared to conventional intramuscular or intradermal injection of the DNA vaccine. Vaccination was well tolerated with mild, transient effects on the skin. This ePatch system is easily portable, without any battery or other power source supply, offering an attractive, inexpensive approach for rapid and accessible DNA vaccination to combat COVID-19, as well as other epidemics.

Interrogating Encapsulated Protein Structure within Metal-Organic Frameworks at Elevated Temperature.

Encapsulating biomacromolecules within metal-organic frameworks (MOFs) can confer thermostability to entrapped guests. It has been hypothesized that the confinement of guest molecules within a rigid MOF scaffold results in heightened stability of the guests, but no direct evidence of this mechanism has been shown. Here, we present a novel analytical method using small-angle X-ray scattering (SAXS) to solve the structure of bovine serum albumin (BSA) while encapsulated within two zeolitic imidazolate frameworks (ZIF-67 and ZIF-8). Our approach comprises subtracting the scaled SAXS spectrum of the ZIF from that of the biocomposite BSA@ZIF to determine the radius of gyration of encapsulated BSA through Guinier, Kratky, and pair distance distribution function analyses. While native BSA exposed to 70 °C became denatured, in situ SAXS analysis showed that encapsulated BSA retained its size and folded state at 70 °C when encapsulated within a ZIF scaffold, suggesting that entrapment within MOF cavities inhibited protein unfolding and thus denaturation. This method of SAXS analysis not only provides insight into biomolecular stabilization in MOFs but may also offer a new approach to study the structure of other conformationally labile molecules in rigid matrices.

Efficient Drug Delivery into Skin Using a Biphasic Dissolvable Microneedle Patch with Water-Insoluble Backing.

Dissolvable microneedle patches (MNPs) enable simplified delivery of therapeutics via the skin. However, most dissolvable MNPs do not deliver their full drug loading to the skin because only some of the drug is localized in the microneedles (MNs), and the rest remains adhered to the patch backing after removal from the skin. In this work, biphasic dissolvable MNPs are developed by mounting water-soluble MNs on a water-insoluble backing layer. These MNPs enable the drug to be contained in the MNs without migrating into the patch backing due to the inability of the drugs to partition into the hydrophobic backing materials during MNP fabrication. In addition, the insoluble backing is poorly wetted upon MN dissolution in the skin, which significantly reduces drug residue on the MNP backing surface after application. These effects enable a drug delivery efficiency of >90% from the MNPs into the skin 5 min after application. This study shows that the biphasic dissolvable MNPs can facilitate efficient drug delivery to the skin, which can improve the accuracy of drug dosing and reduce drug wastage.

Using retinal function to define ischemic exclusion criteria for animal models of glaucoma.

Most animal models of glaucoma rely on induction of ocular hypertension (OHT), yet such models can suffer from high IOPs leading to undesirable retinal ischemia. Thus, animals with IOPs exceeding a threshold (e.g. > 60 mmHg) are often excluded from studies. However, due to the intermittent nature of IOP measurements, this approach may fail to detect ischemia. Conversely, it may also inappropriately eliminate animals with IOP spikes that do not induce ischemic damage. It is known that acute ischemia selectively impairs inner retinal function, which results in a reduced b-wave amplitude. Here, we explore the potential of using electroretinography (ERG) to detect ischemic damage in OHT eyes. 74 Brown Norway rats received a unilateral injection of magnetic microbeads to induce OHT, while contralateral eyes served as controls. IOP was measured every 2-3 days for 14 days after microbead injection. Retinal function was evaluated using dark-adapted bright flash ERG (2.1 log cd•s/m2) prior to, and at 7 and 14 days after, injection. We investigated two criteria for excluding animals: (IOP Criterion) a single IOP measurement > 60 mmHg; or (ERG Criterion) a b-wave amplitude below the 99.5% confidence interval for naïve eyes. 49 of 74 rats passed both criteria, 7 of 74 failed both, and 18 passed one criterion but not the other. We suggest that ERG testing can detect unwelcome ischemic damage in animal models of OHT. Since brief IOP spikes do not necessarily lead to ischemic retinal damage, and because extended periods of elevated IOP can be missed, such ERG-based criteria may provide more objective and robust exclusion criteria in future glaucoma studies.

Optimization of intracellular macromolecule delivery by nanoparticle-mediated photoporation.

Nanoparticle-mediated photoporation is a novel delivery platform for intracellular molecule delivery. We studied the dependence of macromolecular delivery on molecular weight and sought to enhance delivery efficiency. DU145 prostate cancer cells were exposed to pulsed laser beam in the presence of carbon-black nanoparticles. Intracellular uptake of molecules decreased with increasing molecular weight. Attributing this dependence to molecular diffusivity, we hypothesized that macromolecular delivery efficiency could be enhanced by increasing either laser fluence or laser exposure duration at low fluence. We observed increased percentages of macromolecule uptake by cells in both cases. However, trade-off between cell uptake and viability loss was most favorable at low laser fluence (25-29 mJ/cm2) and longer exposure durations (4-5 min). We conclude that long exposure at low laser fluence optimizes intracellular macromolecule delivery by nanoparticle-mediated photoporation, which may be explained by longer time for macromolecules to diffuse into cells, during and between laser pulses.

Mechanisms of sampling interstitial fluid from skin using a microneedle patch.

Although interstitial fluid (ISF) contains biomarkers of physiological significance and medical interest, sampling of ISF for clinical applications has made limited impact due to a lack of simple, clinically useful techniques that collect more than nanoliter volumes of ISF. This study describes experimental and theoretical analysis of ISF transport from skin using microneedle (MN) patches and demonstrates collection of >1 µL of ISF within 20 min in pig cadaver skin and living human subjects using an optimized system. MN patches containing arrays of submillimeter solid, porous, or hollow needles were used to penetrate superficial skin layers and access ISF through micropores (µpores) formed upon insertion. Experimental studies in pig skin found that ISF collection depended on transport mechanism according to the rank order diffusion < capillary action < osmosis < pressure-driven convection, under the conditions studied. These findings were in agreement with independent theoretical modeling that considered transport within skin, across the interface between skin and µpores, and within µpores to the skin surface. This analysis indicated that the rate-limiting step for ISF sampling is transport through the dermis. Based on these studies and other considerations like safety and convenience for future clinical use, we designed an MN patch prototype to sample ISF using suction as the driving force. Using this approach, we collected ISF from human volunteers and identified the presence of biomarkers in the collected ISF. In this way, sampling ISF from skin using an MN patch could enable collection of ISF for use in research and medicine.

Heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice.

Influenza is a persistent threat to public health. Here we report that double-layered peptide nanoparticles induced robust specific immunity and protected mice against heterosubtypic influenza A virus challenges. We fabricated the nanoparticles by desolvating a composite peptide of tandem copies of nucleoprotein epitopes into nanoparticles as cores and cross-linking another composite peptide of four tandem copies of influenza matrix protein 2 ectodomain epitopes to the core surfaces as a coating. Delivering the nanoparticles via dissolvable microneedle patch-based skin vaccination further enhanced the induced immunity. These peptide-only, layered nanoparticles demonstrated a strong antigen depot effect and migrated into spleens and draining (inguinal) lymph nodes for an extended period compared with soluble antigens. This increased antigen-presentation time correlated with the stronger immune responses in the nanoparticle-immunized group. The protection conferred by nanoparticle immunization was transferable by passive immune serum transfusion and depended partially on a functional IgG receptor FcγRIV. Using a conditional cell depletion, we found that CD8+ T cells were involved in the protection. The immunological potency and stability of the layered peptide nanoparticles indicate applications for other peptide-based vaccines and peptide drug delivery.

Feasibility of Hepatitis B Vaccination by Microneedle Patch: Cellular and Humoral Immunity Studies in Rhesus Macaques.

BACKGROUND: This study evaluated dissolvable microneedle patch (dMNP) delivery of hepatitis B vaccine in rhesus macaques and provides evidence that dMNP delivery elicits seroprotective anti-HBs levels comparable with human seroprotection, potentially useful for hepatitis B birth dose vaccination in resource-constrained regions. METHODS: Sixteen macaques were each vaccinated twice; they were treated in 4 groups, with dMNP delivery of AFV at 24 ± 8 µg (n = 4) or 48 ± 14 µg (n = 4), intramuscular injection of AFV (10 µg; n = 4), or intramuscular injection of AAV (10 µg; n = 4). Levels of antibody to hepatitis B surface antigen (HBsAg) (anti-HBs) and HBsAg-specific T-cell responses were analyzed. RESULTS: Six of 8 animals with dMNP delivery of AFV had anti-HBs levels ≥10 mIU/mL after the first vaccine dose. After dMNP delivery of AFV, interferon γ, interleukin 2, and interleukin 4 production by HBsAg-specific T cells was detected. A statistically significant positive correlation was detected between anti-HBs levels and cells producing HBsAg-specific interferon γ and interleukin 2 (T-helper 1-type cytokine) and interleukin 4 (T-helper 2-type cytokine) in all anti-HBs-positive animals. CONCLUSIONS: dMNP delivery of AFV can elicit seroprotective anti-HBs levels in rhesus macaques that are correlated with human seroprotection, and it could be particularly promising for birth dose delivery of hepatitis B vaccine in resource-constrained regions.

Collagenase injection into the suprachoroidal space of the eye to expand drug delivery coverage and increase posterior drug targeting.

Injection into the suprachoroidal space (SCS) allows drug delivery targeted to sclera, choroid, and retina. Here, we studied SCS injection formulated with collagenase to expand drug delivery coverage and increase posterior drug targeting within SCS by breaking down collagen fibrils that link sclera and choroid in the SCS. When 1 µm latex microparticles were injected with a collagenase formulation using microneedles into the SCS of rabbit eyes ex vivo and incubated at 37 °C for 4 h, microparticle delivery coverage increased from 20% to 45% and enhanced posterior drug targeting. Collagenase concentration was optimized to 0.5 mg/mL to maximize expanded posterior delivery and minimize tissue damage. Effects of collagenase injection within SCS increased and then plateaued 4 h after injection. Simultaneous injection of collagenase and microparticles had a greater effect on expanded delivery in the SCS compared to sequential injection. Collagenase injection into the SCS of rabbit eyes in vivo was also effective to expand delivery and was generally well-tolerated, showing transiently lowered IOP, but no apparent lasting adverse effects on ocular tissues such as sclera, choroid, and retina, as determined by analyzing histology, sclera tensile strength, and fundus imaging. We conclude that addition of collagenase during SCS injection can expand drug delivery coverage and increase posterior drug targeting.

Monitoring drug pharmacokinetics and immunologic biomarkers in dermal interstitial fluid using a microneedle patch.

Minimally invasive point-of-care diagnostic devices are of great interest for rapid detection of biomarkers in diverse settings. Although blood is the most common source of biomarkers, interstitial fluid (ISF) is an alternate body fluid that does not clot or contain red blood cells that often complicate analysis. However, ISF is difficult to collect. In this study, we assessed the utility of a microneedle patch to sample microliter volumes of ISF in a simple and minimally invasive manner. We demonstrated the use of ISF collected in this way for therapeutic drug monitoring by showing similar vancomycin pharmacokinetic profiles in ISF and serum from rats. We also measured polio-specific neutralizing antibodies and anti-polio IgG in ISF similar to serum in rats immunized with polio vaccine. These studies demonstrate the potential utility of ISF collected by microneedle patch in therapeutic drug monitoring and immunodiagnostic applications.

Intradermal Vaccination With Adjuvanted Ebola Virus Soluble Glycoprotein Subunit Vaccine by Microneedle Patches Protects Mice Against Lethal Ebola Virus Challenge.

In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.

A Microneedle Patch for Measles and Rubella Vaccination Is Immunogenic and Protective in Infant Rhesus Macaques.

Background: New methods to increase measles and rubella (MR) vaccination coverage are needed to achieve global and regional MR elimination goals. Methods: Here, we developed microneedle (MN) patches designed to administer MR vaccine by minimally trained personnel, leave no biohazardous sharps waste, remove the need for vaccine reconstitution, and provide thermostability outside the cold chain. This study evaluated the immunogenicity of MN patches delivering MR vaccine to infant rhesus macaques. Results: Protective titers of measles neutralizing antibodies (>120 mIU/mL) were detected in 100% of macaques in the MN group and 75% of macaques in the subcutaneous (SC) injection group. Rubella neutralizing antibody titers were >10 IU/mL for all groups. All macaques in the MN group were protected from challenge with wild-type measles virus, whereas 75% were protected in the SC group. However, vaccination by the MN or SC route was unable to generate protective immune responses to measles in infant macaques pretreated with measles immunoglobulin to simulate maternal antibody. Conclusions: These results show, for the first time, that MR vaccine delivered by MN patch generated protective titers of neutralizing antibodies to both measles and rubella in infant rhesus macaques and afforded complete protection from measles virus challenge.

The safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomised, partly blinded, placebo-controlled, phase 1 trial.

BACKGROUND: Microneedle patches provide an alternative to conventional needle-and-syringe immunisation, and potentially offer improved immunogenicity, simplicity, cost-effectiveness, acceptability, and safety. We describe safety, immunogenicity, and acceptability of the first-in-man study on single, dissolvable microneedle patch vaccination against influenza. METHODS: The TIV-MNP 2015 study was a randomised, partly blinded, placebo-controlled, phase 1, clinical trial at Emory University that enrolled non-pregnant, immunocompetent adults from Atlanta, GA, USA, who were aged 18-49 years, naive to the 2014-15 influenza vaccine, and did not have any significant dermatological disorders. Participants were randomly assigned (1:1:1:1) to four groups and received a single dose of inactivated influenza vaccine (fluvirin: 18 µg of haemagglutinin per H1N1 vaccine strain, 17 µg of haemagglutinin per H3N2 vaccine strain, and 15 µg of haemagglutinin per B vaccine strain) (1) by microneedle patch or (2) by intramuscular injection, or received (3) placebo by microneedle patch, all administered by an unmasked health-care worker; or received a single dose of (4) inactivated influenza vaccine by microneedle patch self-administered by study participants. A research pharmacist prepared the randomisation code using a computer-generated randomisation schedule with a block size of 4. Because of the nature of the study, participants were not masked to the type of vaccination method (ie, microneedle patch vs intramuscular injection). Primary safety outcome measures are the incidence of study product-related serious adverse events within 180 days, grade 3 solicited or unsolicited adverse events within 28 days, and solicited injection site and systemic reactogenicity on the day of study product administration through 7 days after administration, and secondary safety outcomes are new-onset chronic illnesses within 180 days and unsolicited adverse events within 28 days, all analysed by intention to treat. Secondary immunogenicity outcomes are antibody titres at day 28 and percentages of seroconversion and seroprotection, all determined by haemagglutination inhibition antibody assay. The trial is completed and registered with ClinicalTrials.gov, number NCT02438423. FINDINGS: Between June 23, 2015, and Sept 25, 2015, 100 participants were enrolled and randomly assigned to a group. There were no treatment-related serious adverse events, no treatment-related unsolicited grade 3 or higher adverse events, and no new-onset chronic illnesses. Among vaccinated groups (vaccine via health-care worker administered microneedle patch or intramuscular injection, or self-administered microneedle patch), overall incidence of solicited adverse events (n=89 vs n=73 vs n=73) and unsolicited adverse events (n=18 vs n=12 vs n=14) were similar. Reactogenicity was mild, transient, and most commonly reported as tenderness (15 [60%] of 25 participants [95% CI 39-79]) and pain (11 [44%] of 25 [24-65]) after intramuscular injection; and as tenderness (33 [66%] of 50 [51-79]), erythema (20 [40%] of 50 [26-55]), and pruritus (41 [82%] of 50 [69-91]) after vaccination by microneedle patch application. The geometric mean titres were similar at day 28 between the microneedle patch administered by a health-care worker versus the intramuscular route for the H1N1 strain (1197 [95% CI 855-1675] vs 997 [703-1415]; p=0·5), the H3N2 strain (287 [192-430] vs 223 [160-312]; p=0·4), and the B strain (126 [86-184] vs 94 [73-122]; p=0·06). Similar geometric mean titres were reported in participants who self-administered the microneedle patch (all p>0·05). The seroconversion percentages were significantly higher at day 28 after microneedle patch vaccination compared with placebo (all p<0·0001) and were similar to intramuscular injection (all p>0·01). INTERPRETATION: Use of dissolvable microneedle patches for influenza vaccination was well tolerated and generated robust antibody responses. FUNDING: National Institutes of Health.

Human Suction Blister Fluid Composition Determined Using High-Resolution Metabolomics.

Interstitial fluid (ISF) surrounds the cells and tissues of the body. Since ISF has molecular components similar to plasma, as well as compounds produced locally in tissues, it may be a valuable source of biomarkers for diagnostics and monitoring. However, there has not been a comprehensive study to determine the metabolite composition of ISF and to compare it to plasma. In this study, the metabolome of suction blister fluid (SBF), which largely consists of ISF, collected from 10 human volunteers was analyzed using untargeted high-resolution metabolomics (HRM). A wide range of metabolites were detected in SBF, including amino acids, lipids, nucleotides, and compounds of exogenous origin. Various systemic and skin-derived metabolite biomarkers were elevated or found uniquely in SBF, and many other metabolites of clinical and physiological significance were well correlated between SBF and plasma. In sum, using untargeted HRM profiling, this study shows that SBF can be a valuable source of information about metabolites relevant to human health.

Effect of laser fluence, nanoparticle concentration and total energy input per cell on photoporation of cells.

Intracellular delivery of molecules can be increased by laser-exposure of carbon black nanoparticles to cause photoporation of the cells. Here we sought to determine effects of multiple laser exposure parameters on intracellular uptake and cell viability with the goal of determining a single unifying parameter that predicts cellular bioeffects. DU145 human prostate cancer cells in suspension with nanoparticles were exposed to near-infrared nanosecond laser pulses over a range of experimental conditions. Increased bioeffects (i.e., uptake and viability loss determined by flow cytometry) were seen when increasing laser fluence, number of pulses and nanoparticle concentration, and decreasing cell concentration. Bioeffects caused by different combinations of these four parameters were generally predicted by their cumulative energy input per cell, which served as a unifying parameter. This indicates that photoporation depends on what appears to be the cumulative effect of multiple cell-nanoparticle interactions from neighboring nanoparticles during a series of laser pulses.

Energy Transfer Mechanisms during Molecular Delivery to Cells by Laser-Activated Carbon Nanoparticles.

Previous studies have shown that exposure of carbon black nanoparticles to nanosecond pulsed near-infrared laser causes intracellular delivery of molecules through hypothesized transient breaks in the cell membrane. The goal of this study is to determine the underlying mechanisms of sequential energy transfer from laser light to nanoparticle to fluid medium to cell. We found that laser pulses on a timescale of 10 ns rapidly heat carbon nanoparticles to temperatures on the order of 1200 K. Heat is transferred from the nanoparticles to the surrounding aqueous medium on a similar timescale, causing vaporization of the surrounding water and generation of acoustic emissions. Nearby cells can be impacted thermally by the hot bubbles and mechanically by fluid mechanical forces to transiently increase cell membrane permeability. The experimental and theoretical results indicate that transfer of momentum and/or heat from the bubbles to the cells are the dominant mechanisms of energy transfer that results in intracellular uptake of molecules. We further conclude that neither thermal expansion of the nanoparticles nor a carbon-steam chemical reaction play a significant role in the observed effects on cells, and that acoustic pressure appears to be concurrent with, but not essential to, the observed bioeffects.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-ß-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Distribution of particles, small molecules and polymeric formulation excipients in the suprachoroidal space after microneedle injection.

The purpose of this work was to determine the effect of injection volume, formulation composition, and time on circumferential spread of particles, small molecules, and polymeric formulation excipients in the suprachoroidal space (SCS) after microneedle injection into New Zealand White rabbit eyes ex vivo and in vivo. Microneedle injections of 25-150 µL Hank's Balanced Salt Solution (HBSS) containing 0.2 µm red-fluorescent particles and a model small molecule (fluorescein) were performed in rabbit eyes ex vivo, and visualized via flat mount. Particles with diameters of 0.02-2 µm were co-injected into SCS in vivo with fluorescein or a polymeric formulation excipient: fluorescein isothiocyanate (FITC)-labeled Discovisc or FITC-labeled carboxymethyl cellulose (CMC). Fluorescent fundus images were acquired over time to determine area of particle, fluorescein, and polymeric formulation excipient spread, as well as their co-localization. We found that fluorescein covered a significantly larger area than co-injected particles when suspended in HBSS, and that this difference was present from 3 min post-injection onwards. We further showed that there was no difference in initial area covered by FITC-Discovisc and particles; the transport time (i.e., the time until the FITC-Discovisc and particle area began dissociating) was 2 d. There was also no difference in initial area covered by FITC-CMC and particles; the transport time in FITC-CMC was 4 d. We also found that particle size (20 nm-2 µm) had no effect on spreading area when delivered in HBSS or Discovisc. We conclude that (i) the area of particle spread in SCS during injection generally increased with increasing injection volume, was unaffected by particle size, and was significantly less than the area of fluorescein spread, (ii) particles suspended in low-viscosity HBSS formulation were entrapped in the SCS after injection, whereas fluorescein was not and (iii) particles co-injected with viscous polymeric formulation excipients co-localized near the site of injection in the SCS, continued to co-localize while spreading over larger areas for 2-4 days, and then no longer co-localized as the polymeric formulation excipients were cleared within 1-3 weeks and the particles remained largely in place. These data suggest that particles encounter greater barriers to flow in SCS compared to molecules and that co-localization of particles and polymeric formulation excipients allows spreading over larger areas of the SCS until the particles and excipients dissociate.