Fundamental role for the creatine kinase pathway in protection from murine colitis.

Inflammatory diseases of the digestive tract, including inflammatory bowel disease, cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in inflammatory bowel disease patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CK brain type/CK mitochondrial form (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length, and histology). In a broad cytokine profiling, CKdKO mice expressed near absent interferon gamma (IFN-γ) levels. We identified losses in IFN-γ production from CD4+ and CD8+ T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. Extensions of these studies identified basal stabilization of the transcription factor hypoxia-inducible factor in CKdKO splenocytes and pharmacological stabilization of hypoxia-inducible factor resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4+ and CD8+ T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

Fundamental role for the creatine kinase pathway in protection from murine colitis.

Inflammatory diseases of the digestive tract, including inflammatory bowel disease (IBD), cause metabolic stress within mucosal tissue. Creatine is a key energetic regulator. We previously reported a loss of creatine kinases (CKs) and the creatine transporter expression in IBD patient intestinal biopsy samples and that creatine supplementation was protective in a dextran sulfate sodium (DSS) colitis mouse model. In the present studies, we evaluated the role of CK loss in active inflammation using the DSS colitis model. Mice lacking expression of CKB/CKMit (CKdKO) showed increased susceptibility to DSS colitis (weight loss, disease activity, permeability, colon length and histology). In a broad cytokine profiling, CKdKO mice expressed near absent IFN-γ levels. We identified losses in IFN-γ production from CD4 + and CD8 + T cells isolated from CKdKO mice. Addback of IFN-γ during DSS treatment resulted in partial protection for CKdKO mice. We identified basal stabilization of the transcription factor hypoxia-inducible factor (HIF) in CKdKO splenocytes and pharmacological stabilization of HIF resulted in reduced IFN-γ production by control splenocytes. Thus, the loss of IFN-γ production by CD4 + and CD8 + T cells in CKdKO mice resulted in increased colitis susceptibility and indicates that CK is protective in active mucosal inflammation.

The TNFΔARE Mouse as a Model of Intestinal Fibrosis.

Crohn disease (CD) is a highly morbid chronic inflammatory disease. Although many patients with CD also develop fibrostenosing complications, there are no medical therapies for intestinal fibrosis. This is due, in part, to a lack of high-fidelity biomimetic models to enhance understanding and drug development, which highlights the need for developing in vivo models of inflammatory bowel disease-related intestinal fibrosis. This study investigates whether the TNFΔARE mouse, a model of ileal inflammation, also develops intestinal fibrosis. Several clinically relevant outcomes were studied, including features of structural fibrosis, histologic fibrosis, and gene expression. These include the use of a new luminal casting technique, traditional histologic outcomes, use of second harmonic imaging, and quantitative PCR. These features were studied in aged TNFΔARE mice as well as in cohorts of numerous ages. At >24 weeks of age, TNFΔARE mice developed structural, histologic, and transcriptional changes of ileal fibrosis. Protein and RNA expression profiles showed changes as early as 6 weeks, coinciding with histologic changes as early as 14 to 15 weeks. Overt structural fibrosis was delayed until at least 16 weeks and was most developed after 24 weeks. This study found that the TNFΔARE mouse is a viable and highly tractable model of ileal fibrosis. This model and the techniques used herein can be leveraged for both mechanistic studies and therapeutic development for the treatment of intestinal fibrosis.

Women Living with Human Immunodeficiency Virus with Intended and Unintended Pregnancy: Characteristics and Patient Outcomes.

Background: Rates of unintended pregnancy may be higher in women living with human immunodeficiency virus (WLWH) than in the general population, and it is unclear how populations of WLWH with intended and unintended pregnancy differ. We compared baseline characteristics and outcomes between WLWH with intended and unintended pregnancy. Materials and Methods: We conducted a retrospective analysis of WLWH enrolled in a human immunodeficiency virus (HIV) and Pregnancy clinic from 2003 to 2014. Data were analyzed using descriptive statistics, chi-square test, Student's t-test, one-way analysis of variance, and linear and logistic regression analysis. Two-tailed p-value <0.05 was considered significant. The study was approved by the Johns Hopkins University School of Medicine Institutional Review Board. Results: Sixty-nine (27.1%) of 255 women reported an intended pregnancy. Women with intended pregnancy (WWIP) were more likely to be older, White, married, privately insured, and college educated. WWIP were less likely to use tobacco (15.9% vs. 44.2%, p < 0.001), alcohol (2.9% vs. 11.1%, p = 0.041), opiates (0.0% vs. 19.3%, p < 0.001), or cocaine (2.9% vs. 21.0%, p < 0.001) during pregnancy, more likely to disclose their HIV status to the father of the baby by delivery (100.0% vs. 15.8%, p < 0.001), and more likely to receive less effective contraception at delivery (condoms 14.9% vs. 4.8%, p = 0.024; sterilization 11.9% vs. 22.1%, p = 0.028). In multivariate regression analysis, pregnancy intendedness was an important predictor of nondetectable viral load at pregnancy entry but not at delivery. Conclusions: WLWH vary in their baseline characteristics and pregnancy outcomes depending on pregnancy intendedness, highlighting the need to improve pregnancy timing in WLWH and intensify interventions for women with unintended pregnancy.

Pregnancy in an Urban Cohort of Adolescents Living with Human Immunodeficiency Virus: Characteristics and Outcomes in Comparison to Adults.

Adolescents (13-24 years of age) account for more than one-fifth of new HIV diagnoses yearly, and the United States has one of the highest rates of adolescent pregnancy among high resource countries. However, there is limited information on the characteristics and outcomes of adolescents living with HIV (ALWHIV) during pregnancy and differences with pregnancy in adults living with HIV. We performed a retrospective cohort study to compare demographic characteristics, HIV viral suppression, and pregnancy outcomes in adolescents (n = 90) as compared with adults (n = 250) in an urban HIV pregnancy clinic from 2003 to 2015. Seventy-one women overall were diagnosed with HIV during pregnancy (adolescents, 25/90; adults, 46/250). One-fifth of adolescents acquired HIV perinatally. Adolescents were more likely than adults to have unintended pregnancy (83.6% vs. 68.7%, p = 0.016) and were less likely to be virally suppressed at delivery (50.0% vs. 69.7% overall, p = 0.001; 48.0% vs. 78.2% in postuniversal antiretroviral therapy era, p = 0.007%). Over one-third of adolescents reported a history of any illicit substance use, and adolescents were more likely than adults to use marijuana during pregnancy (29.2% vs. 16.9%, p = 0.013). Adolescents were less likely to experience preterm labor (11.0% vs. 24.1%, p = 0.012) or preterm premature rupture of membranes (3.7% vs. 16.7%, p = 0.003). There was one case of maternal-fetal transmission, which occurred in an adult pregnancy. Despite the high rate of unintended pregnancy, one-third of adolescents were discharged without an identified contraception plan. We identify several opportunities for intervention to improve reproductive health outcomes in ALWHIV.

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells.

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

Cushing's Syndrome in a Pregnant Patient Secondary to Adrenal Adenoma: Treatment With Unilateral Adrenalectomy.

BACKGROUND: A pregnant woman presented with Cushing's syndrome (CS) secondary to adrenal adenoma and was treated with laparoscopic-assisted right adrenalectomy during the second trimester of pregnancy. SUMMARY: Our patient is a 33-year-old woman who presented with hypertension and cushingoid appearance at 21 weeks gestational age. Laboratory evidence indicated CS was likely, and imaging found a 2.3 x 3.0 cm right adrenal nodule as a suggested cause. Laparoscopic-assisted right adrenalectomy was performed at 23 weeks 4 days gestation without complication. Pathology of the removed specimen revealed an adrenal gland containing a 3.0 x 3.0 x 2.0 cm well-circumscribed proliferation of adrenal cortical cells without significant atypia. This report describes the rare occurrence of CS during pregnancy and subsequent successful surgical management. We review the diagnosis of CS during pregnancy and surgical considerations. CONCLUSION: CS, while a rare entity in the general population, is even more unique in the setting of pregnancy due to the negative effects of hypercortisolism on fertility. CS during pregnancy presents a diagnostically complicated scenario, along with specific considerations necessary for successful surgical management.

Is TNF-α Inhibitor-Induced Psoriasiform Dermatitis Always Psoriasiform?

Tumor necrosis factor-α inhibitors (TNFIs) have significantly improved the quality of life for patients with psoriasis and psoriatic arthritis. Despite their beneficial effects, TNFIs have been reported to cause paradoxical 'psoriasiform' eruptions.1 Although this nomenclature has become pervasive in the dermatology lexicon, there is a growing body of literature highlighting the protean clinical presentation of this eruption (Figure 1), which could ultimately lead to a delayed diagnosis.2-5 The diversity of the morphology highlights the importance of identifying key histopathologic characteristics, which to date have not been well-characterized.2

X-Linked Huwe1 Is Essential for Oocyte Maturation and Preimplantation Embryo Development.

HUWE1 is a HECT-domain ubiquitin E3 ligase expressed in various tissues. Although HUWE1 is known to promote degradation of the tumor suppressor p53, given a growing list of its substrates, in vivo functions of HUWE1 remain elusive. Here, we investigated the role of HUWE1 in the female reproductive system. Homozygous deletion of Huwe1 in mouse oocytes of primary follicles caused oocyte death and female infertility, whereas acute depletion of HUWE1 protein by Trim-Away technology did not impact oocytes from antral follicles. Interestingly, oocytes from Huwe1 heterozygous females matured and fertilized normally, but the majority of embryos that lacked maternal Huwe1 were arrested at the morula stage after fertilization. Consequently, Huwe1 heterozygous females only produced wild-type pups. Concomitant knockout of p53 did not recover fertility of the Huwe1 knockout females. These findings make HUWE1 a unique and critical maternal factor indispensable for maintaining the quality of oocytes and embryos.

Creatine Transporter, Reduced in Colon Tissues From Patients With Inflammatory Bowel Diseases, Regulates Energy Balance in Intestinal Epithelial Cells, Epithelial Integrity, and Barrier Function.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs) have intestinal barrier dysfunction. Creatine regulates energy distribution within cells and reduces the severity of colitis in mice. We studied the functions of the creatine transporter solute carrier family 6 member 8 (SLC6A8, also called CRT) in intestinal epithelial cells (IECs) and mice, and we measured levels in mucosal biopsies from patients with IBD. METHODS: Colon biopsy specimens from patients with IBD (30 with Crohn's disease and 27 with ulcerative colitis) and 30 patients without IBD (control individuals) and colon tissues from mice (with and without disruption of Crt) were analyzed by immunofluorescence, immunoblots, and/or quantitative reverse-transcription polymerase chain reaction (qRT-PCR). CRT was knocked down or overexpressed in T84 cells, which were analyzed by immunofluorescence, immunoblots, high-performance liquid chromatography (to measure creatine levels), qRT-PCR, transepithelial electrical resistance, barrier function, actin localization, wound healing, mitochondrial oxygen consumption, and glycolysis extracellular acidification rate assays. Organoids from colon cells of CRT-knockout mice and control mice were analyzed by qRT-PCR, immunoblot, and transepithelial electrical resistance. RESULTS: CRT localized around tight junctions (TJs) of T84 IECs. In analyses of IECs with CRT knockdown or overexpression, we found that CRT regulates intracellular creatine, barrier formation, and wound healing. CRT-knockout organoids also had diminished barrier formation. In the absence of adequate creatine, IECs transition toward a stressed, glycolysis-predominant form of metabolism; this resulted in leaky TJs and mislocalization of actin and TJ proteins. Colon tissues from patients with IBD had reduced levels of CRT messenger RNA compared with those from control individuals. CONCLUSIONS: In an analysis of IEC cell lines and colonoids derived from CRT-knockout mice, we found that CRT regulates energy balance in IECs and thereby epithelial integrity and barrier function. Mucosal biopsy specimens from patients with ulcerative colitis and inactive Crohn's disease have lower levels of CRT, which might contribute to the reduced barrier function observed in patients with IBD.

Delivery of thymoquinone to cancer cells with as1411-conjugated nanodroplets.

Systemic delivery of conventional chemotherapies can cause negative systemic toxicity, including reduced immunity and damage to organs such as the heart and kidneys-limiting the maximum dose that can be administered. Targeted therapies appear to address this problem by having a specific target while mitigating off-target effects. Biocompatible perfluorocarbon-based nanodroplet emulsions encapsulated by a phospholipid shell are in development for delivery of molecular compounds and hold promise as vehicles for targeted delivery of chemotherapeutics to tumors. When ultrasound is applied, perfluorocarbon will undergo a phase change-ultimately inducing transient perforation of the cell membrane when in close proximity, which is more commonly known as "sonoporation." Sonoporation allows enhanced intracellular delivery of molecular compounds and will reseal to encapsulate the molecular compound intracellularly. In this study, we investigated delivery of thymoquinone (TQ), a natural hydrophobic phytochemical compound with bioactivity in cancer cells. In addition, we conjugated a G-quadruplex aptamer, 'AS1411', to TQ-loaded nanodroplets and explored their effects on multiple human cancer cell lines. AS1411 binds nucleolin, which is over-expressed on the surface of cancer cells, and in addition to its tumor-targeting properties AS1411 has also been shown to induce anti-cancer effects. Thymoquinone was loaded onto AS1411-conjugated nanodroplet emulsion to assess activity against cancer cells. Confocal microscopy indicated uptake of AS1411-conjugated nanodroplets by cancer cells. Furthermore, AS1411-conjugated nanoemulsions loaded with TQ significantly enhanced cytotoxicity in cancer cells compared to free compound. These results demonstrate that AS1411 can be conjugated onto nanodroplet emulsions for targeted delivery to human cancer cells. This novel formulation offers significant potential for targeted delivery of hydrophobic chemotherapeutics to tumors for cancer treatment.

Hydrogen Peroxide Topical Solution, 40% (w/w) for the Treatment of Seborrheic Keratoses: A Review

HP40 (Eskata™) is a stabilized, topical solution of 40% hydrogen peroxide (H2O2) packaged in an applicator pen that is US FDA-approved to treat seborrheic keratoses (SKs). By harnessing the oxidative capabilities of H2O2 , 1-2 treatments with HP40 produced a higher rate of clearance of four SKs per patient compared to vehicle in two phase 3 trials. The clearance rate was higher for the face than the trunk and extremities. Similarly, the risks of pigmentary changes and scarring from HP40 were lower for the face than other locations. Further, based on an ex vivo study, HP40 may be less cytotoxic to melanocytes than cryotherapy, but clinical trials comparing these therapies are needed. Limitations of HP40 are its low efficacy and requirement of multiple treatments, which can result in elevated costs. The application can also be time-consuming, though extenders or even staff members can apply it. Therefore, HP40 may be better reserved for the treatment of facial SKs.

Clinical Experience With 40% Hydrogen Peroxide Topical Solution for the Treatment of Seborrheic Keratosis

Despite reassurances about the benign nature of seborrheic keratoses (SKs), patients often request treatment due to cosmetic concerns or for symptomatic relief when SKs become irritated or pruritic. Treatment options include cryotherapy, surgical techniques, and topical therapies. In this study, we present two patients with SKs located on their face and neck who received in-office treatment with 40% Hydrogen Peroxide Topical Solution (Eskata™, HP40), a new FDA-approved topical therapy that has demonstrated efficacy in phase 3 trials. Compared to non-topical, more invasive techniques, HP40 may lead to less pigmentary changes, and may be more efficacious for SKs on the face and neck. Both patients received two treatment courses of HP40, which resulted in positive therapeutic outcomes, including the absence of scarring and pigmentary changes. In addition to the case presentations, we will discuss considerations for appropriate administration of HP40 to maximize clinical outcomes. J Drugs Dermatol. 2019;18(7 Suppl):s173-177.

A Survey-Based Comparison of Sun Safety Practices in a Representative Cohort of the General Public Versus Attendees of a Skin Cancer Screening

A large proportion of data on photoprotective practices is yielded from free skin cancer screenings. However, the sun safety practices of populations who seek these skin cancer screenings may differ from the general public. To examine differences in skin cancer prevention practices and risk factors, we surveyed pedestrians at six locations in Washington, DC (public group, n=285) and attendees of a free skin cancer screening (screening group, n=144) using an IRB-approved survey. The screening group was older and included more individuals with fair skin than the public group. Respondents from the screening group were significantly more likely to always wear sunscreen, always seeks shade, and always or sometimes wear sun-protective clothing than the public group (P<0.05). To examine whether younger and non-white participants, who were less likely to attend our free screening, have different practices and risk factors than older and white participants, respectively, we compared survey answers for all participants by age and race. White participants were more likely to always or sometimes wear sunscreen and sun-protective clothing than non-white participants (P<0.05). Patients over 61 years were more likely to always seek shade and wear sun-protective clothing than those younger than 31 years (P<0.05). Therefore, free skin cancer screenings need to be better popularized among non-white and younger populations or more effective educational vehicles are needed. J Drugs Dermatol. 2019;18(7):649-653.

A Head-to-Head Comparison of Topical Collagen Powder to Primary Closure for Acute Full-Thickness Punch Biopsy-Induced Human Wounds: An Internally Controlled Pilot Study

Background: Collagen-based products have been implemented in wound healing due to collagen's hemostatic properties, low antigenicity, and poor culture ability. Objective: To compare the rate and quality of full-thickness wound healing for topical collagen powder and primary closure. Methods: Eight volunteers received one 4 mm punch biopsy on each thigh. One wound was managed with primary closure while the other received daily collagen powder. Wounds were biopsied at four weeks for histopathological analysis. Subjects rated itch, pain, and treatment preferences at weeks 1, 2, 4, 6, and 12. Results: Six out of eight collagen-treated wounds were completely healed 4 weeks after initial wounding. Histologic analysis of the wounds revealed epidermal re-epithelization in both groups. More organized granulation tissue was noted in collagen-treated wounds and confirmed using Masson trichrome and CD31 staining for collagen and neoangiogenesis, respectively. Subjects reported similar itch and pain metrics between wounds. Both subjects and blinded dermatologists preferred the early cosmetic appearance of collagen-treated wounds over primarily closed wounds. Limitations: Small sample size, absence of negative control. Conclusion: These data suggest that collagen powder is non-inferior to primary closure at the macro- and microscopic levels, while possibly leading to superior early cosmetic outcomes and accelerated histologic wound maturation. Ethics/Clinical Trials Registration: Study was approved by the George Washington University Institutional Review Board (IRB protocol #121745). ClinicalTrials.gov: NCT03481907. J Drugs Dermatol. 2019;18(7):667-673.

A Survey-Based Comparison of Sun Safety Practices in a Representative Cohort of the General Public Versus Attendees of a Skin Cancer Screening

A large proportion of data on photoprotective practices is yielded from free skin cancer screenings. However, the sun safety practices of populations who seek these skin cancer screenings may differ from the general public. To examine differences in skin cancer prevention practices and risk factors, we surveyed pedestrians at six locations in Washington, DC (public group, n=285) and attendees of a free skin cancer screening (screening group, n=144) using an IRB-approved survey. The screening group was older and included more individuals with fair skin than the public group. Respondents from the screening group were significantly more likely to always wear sunscreen, always seeks shade, and always or sometimes wear sun-protective clothing than the public group (P<0.05). To examine whether younger and non-white participants, who were less likely to attend our free screening, have different practices and risk factors than older and white participants, respectively, we compared survey answers for all participants by age and race. White participants were more likely to always or sometimes wear sunscreen and sun-protective clothing than non-white participants (P<0.05). Patients over 61 years were more likely to always seek shade and wear sun-protective clothing than those younger than 31 years (P<0.05). Therefore, free skin cancer screenings need to be better popularized among non-white and younger populations or more effective educational vehicles are needed. J Drugs Dermatol. 2019;18(7):649-653.

Hydrogen peroxide and cutaneous biology: Translational applications, benefits, and risks.

Hydrogen peroxide (H2O2) is an endogenous reactive oxygen species that contributes to oxidative stress directly as a molecular oxidant and indirectly through free radical generation. Topically applied 1% to 45% H2O2 can be used for a range of clinical purposes, which will be reviewed here in addition to its safety. In concentrations from 1% to 6%, H2O2 has antimicrobial properties and can act as a debriding agent through its effervescence, making low-concentration H2O2 useful for wound care. H2O2 has also been shown to promote venous insufficiency ulcer healing, but studies in other wound types are needed. In 1% formulations, H2O2 is used outside the United States to treat acne and has shown efficacy similar to or greater than benzoyl peroxide, with reduced side effects. In a concentration of 40%, H2O2 is US Food and Drug Administration-approved to treat seborrheic keratoses and may cause fewer pigmentary changes than cryotherapy, although elimination often requires 2 to 4 treatments. However, H2O2 should be used with caution, as exposure can cause adverse effects through its oxidant capabilities. Low H2O2 concentrations cause only transient symptoms (blanching and blistering), but exposure to 9% to 45% H2O2 can cause more severe skin damage, including epidermal necrosis leading to erythema and bullae. Overall, H2O2 has numerous therapeutic uses, and novel indications, such as treating actinic keratoses and skin cancers, continue to be explored.

Cell Type-Specific p38δ Targeting Reveals a Context-, Stage-, and Sex-Dependent Regulation of Skin Carcinogenesis.

Activation and/or upregulated expression of p38δ are demonstrated in human skin malignancies including cutaneous squamous cell carcinoma, suggesting a role for p38δ in skin carcinogenesis. We previously reported that mice with germline deletion of the p38δ gene are significantly protected from chemical skin carcinogenesis. Here, we investigated the effects of cell-selective targeted ablation of p38δ in keratinocytes and in immune (myeloid) cells on skin tumor development in a two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical mouse skin carcinogenesis model. Conditional keratinocyte-specific p38δ ablation (p38δ-cKO∆K) did not influence the latency, incidence, or multiplicity of chemically-induced skin tumors, but led to increased tumor volume in females during the TPA promotion stage, and reduced malignant progression in males and females relative to their wild-type counterparts. In contrast, conditional myeloid cell-specific p38δ deletion (p38δ-cKO∆M) inhibited DMBA/TPA-induced skin tumorigenesis in male but not female mice. Thus, tumor onset was delayed, and tumor incidence, multiplicity, and volume were reduced in p38δ-cKO∆M males compared with control wild-type males. Moreover, the percentage of male mice with malignant tumors was decreased in the p38δ-cKO∆M group relative to their wild-type counterparts. Collectively, these results reveal that cell-specific p38δ targeting modifies susceptibility to chemical skin carcinogenesis in a context-, stage-, and sex-specific manner.

Effect of Molecular Weight on Sonoporation-Mediated Uptake in Human Cells.

Ultrasound-induced microbubble destruction can enhance drug delivery to cells. The molecular weight of therapeutic compounds varies significantly (from <1 kDa for small molecule drugs, to 7-15 kDa for siRNAs/miRNAs, to >1000 kDa for DNA plasmids). Therefore, the objective of this study was to determine the relationship between uptake efficiency and molecular weight using equal molar concentrations. Uptake efficiency of fluorescent compounds with different molecular weights (0.3, 10 and 2000 kDa) was explored in vitro using human cardiac mesenchymal cells and breast cancer cells exposed to microbubbles and 2.5-MHz ultrasound pulses. Uptake by viable cells was quantified using flow cytometry. After correction for the fluorescence yield of each compound, there was a significant size-dependent difference in fluorescence intensity, indicating an inverse relationship between size and uptake efficiency. These results suggest that diffusion of therapeutic compounds across permeabilized cell membranes may be an important mechanism for ultrasound-mediated drug delivery.