Intracellular Cargo Delivery Research Articles

Targeted intracellular delivery of molecular cargo to hypoxic human breast cancer stem cells.

Cancer stem cells (CSCs) drive tumorigenesis, are responsible for metastasis, and resist conventional therapies thus posing significant treatment challenges. CSCs reside in hypoxic tumor regions and therefore, effective therapies must target CSCs within this specific microenvironment. CSCs are characterized by limited distinguishable features, however, surface displayed phosphatidylserine (PS) appears to be characteristic of stem cells and offers a potential target. GlaS, a truncated coagulation protein that is internalized after binding PS, was investigated for intracellular delivery of molecular payloads to CSCs. Intracellular delivery via GlaS was enhanced in patient-derived CD44+ mammary CSCs under hypoxic conditions relative to physoxia or hyperoxia. In vivo, GlaS successfully targeted hypoxic tumor regions, and functional delivery of molecular cargo was confirmed using luciferin conjugated to GlaS via a disulfide linkage (GlaS-SS-luc), which releases luciferin upon intracellular glutathione reduction. Bioluminescence imaging demonstrated effective GlaS-mediated delivery of luciferin, a model drug, to CSCs in culture and in vivo. These findings offer the promise of directed delivery of therapeutic agents to intracellular targets in CSCs.

Artificial Internalizing Receptors: Intracellular Delivery of Cargo Through Bio-Orthogonal Recognition.

Drug targeting is a methodology that helps to overcome the side effects of therapeutic molecules. However, insufficient targeting specificity and the on-target/off-site delivery leave much room for improvement in the targeting endeavors. One approach to enhance the specificity of drug targeting is to engineer artificial receptors with recognition ligands not observed in nature. To this end, artificial internalizing receptors that feature cholesterylamine as the artificial pull-in mechanism, and an anti-fluorescein antibody as the exofacial recognition and capture tool are developed. Fluorescein labeling is among the most routine techniques in biochemistry and can readily provide a way to make cognate derivatives for receptor-mediated endocytosis using these artificial receptors. Herein, the synthesis and the structure-activity relationship for these artificial receptors are detailed, their potency and efficacy in mediating drug delivery for the antibody-drug conjugates are illustrated, and the scope and limitations of targeting the chemically engineered cells via artificial receptors are investigated. Taken together, the presented data explore an innovative approach to drug targeting and contribute to the development of techniques in cell engineering using the tools of chemistry.

Characterizing Extracellular Vesicles Generated from the Integra CELLine Culture System and Their Endocytic Pathways for Intracellular Drug Delivery.

Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 1010 EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery.

Timescale dependent homogeneous assembly of lipid-polymer hybrid nanoparticles in laminar mixing for enhanced lung cancer treatment

The homogeneity of core–shell structure lipid-polymer hybrid nanoparticles (HNPs) is crucial for efficient intracellular cargo delivery. While microfluidic techniques have been recognized for their capabilities in precisely controlling the size and drug encapsulation efficiency, there is little research exploring the impact of mass transfer behavior within microchannels on the homogeneous assembly process of HNPs. Here, we manipulated the laminar flow state within TrM device to achieve controlled assembly of HNPs and explored a timescale dependent assembly technique to ensure the homogeneity of folate modified HNPs (FHNPs). Our founding indicate that this method enabled the control over the growth kinetics of PLGA cores within a timescale (τgrow) of 1–15 ms. The homogeneous ligand assembly on the surface of FHNPs can only be achieved when τgrow exceeds the timescale of functional lipid coating (τcoating). Compared to conventional bulk mixing methods, this strategy significantly reduces the risk of heterogeneous assembly in FHNPs fabrication and ensures consistent reproducibility across batches. The homogeneous FHNPs (HFHNPs) fabricated through this method exhibit precise control over particle size (ranging from 75 nm to 150 nm), low polydispersity index, and consistent core–shell structure. Furthermore, this strategy enhances the density of available folate ligands on the surface of FHNPs. In subsequent in vitro and in vivo anti-tumor assays, the icariside Ⅱ (IS) loaded HFHNPs (IS@HFHNPs) exhibited enhanced cellular uptake and tumor accumulation behavior. the timescale dependent homogeneous assembly method for HNPs developed in this research presents a promising avenue for maintaining quality in the synthesis of self-assembly composite nanomecidine.

Plasma membrane damage limits cytoplasmic delivery by conventional cell penetrating peptides.

Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.

3D Computational Modeling of Defective Early Endosome Distribution in Human iPSC-Based Cardiomyopathy Models.

Intracellular cargo delivery via distinct transport routes relies on vesicle carriers. A key trafficking route distributes cargo taken up by clathrin-mediated endocytosis (CME) via early endosomes. The highly dynamic nature of the endosome network presents a challenge for its quantitative analysis, and theoretical modelling approaches can assist in elucidating the organization of the endosome trafficking system. Here, we introduce a new computational modelling approach for assessment of endosome distributions. We employed a model of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with inherited mutations causing dilated cardiomyopathy (DCM). In this model, vesicle distribution is defective due to impaired CME-dependent signaling, resulting in plasma membrane-localized early endosomes. We recapitulated this in iPSC-CMs carrying two different mutations, TPM1-L185F and TnT-R141W (MUT), using 3D confocal imaging as well as super-resolution STED microscopy. We computed scaled distance distributions of EEA1-positive vesicles based on a spherical approximation of the cell. Employing this approach, 3D spherical modelling identified a bi-modal segregation of early endosome populations in MUT iPSC-CMs, compared to WT controls. Moreover, spherical modelling confirmed reversion of the bi-modal vesicle localization in RhoA II-treated MUT iPSC-CMs. This reflects restored, homogeneous distribution of early endosomes within MUT iPSC-CMs following rescue of CME-dependent signaling via RhoA II-dependent RhoA activation. Overall, our approach enables assessment of early endosome distribution in cell-based disease models. This new method may provide further insight into the dynamics of endosome networks in different physiological scenarios.

Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli invitro and in the brain of mice invivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Exosome-coated oxygen nanobubble-laden hydrogel augments intracellular delivery of exosomes for enhanced wound healing

Wound healing is an obvious clinical concern that can be hindered by inadequate angiogenesis, inflammation, and chronic hypoxia. While exosomes derived from adipose tissue-derived stem cells have shown promise in accelerating healing by carrying therapeutic growth factors and microRNAs, intracellular cargo delivery is compromised in hypoxic tissues due to activated hypoxia-induced endocytic recycling. To address this challenge, we have developed a strategy to coat oxygen nanobubbles with exosomes and incorporate them into a polyvinyl alcohol/gelatin hybrid hydrogel. This approach not only alleviates wound hypoxia but also offers an efficient means of delivering exosome-coated nanoparticles in hypoxic conditions. The self-healing properties of the hydrogel, along with its component, gelatin, aids in hemostasis, while its crosslinking bonds facilitate hydrogen peroxide decomposition, to ameliorate wound inflammation. Here, we show the potential of this multifunctional hydrogel for enhanced healing, promoting angiogenesis, facilitating exosome delivery, mitigating hypoxia, and inhibiting inflammation in a male rat full-thickness wound model.

Targeted intracellular delivery of dimeric STINGa by two pHLIP peptides for treatment of solid tumors.

Introduction: We have developed a delivery approach that uses two pHLIP peptides that collaborate in the targeted intracellular delivery of a single payload, dimeric STINGa (dMSA). Methods: dMSA was conjugated with two pHLIP peptides via S-S cleavable self-immolating linkers to form 2pHLIP-dMSA. Results: Biophysical studies were carried out to confirm pH-triggered interactions of the 2pHLIP-dMSA with membrane lipid bilayers. The kinetics of linker self-immolation and dMSA release, the pharmacokinetics, the binding to plasma proteins, the stability of the agent in plasma, the targeting and resulting cytokine activation in tumors, and the biodistribution of the construct was investigated. This is the first study demonstrating that combining the energy of the membrane-associated folding of two pHLIPs can be utilized to enhance the targeted intracellular delivery of large therapeutic cargo payloads. Discussion: Linking two pHLIPs to the cargo extends blood half-life, and targeted delivery of dimeric STINGa induces tumor eradication and the development of robust anti-cancer immunity.

Uptake pathways of cell-penetrating peptides in the context of drug delivery, gene therapy, and vaccine development

Cell-penetrating peptides have been extensively utilized for the purpose of facilitating the intracellular delivery of cargo that is impermeable to the cell membrane. The researchers have exhibited proficient delivery capabilities for oligonucleotides, thereby establishing cell-penetrating peptides as a potent instrument in the field of gene therapy. Furthermore, they have demonstrated a high level of efficiency in delivering several additional payloads. Cell penetrating peptides (CPPs) possess the capability to efficiently transport therapeutic molecules to specific cells, hence offering potential remedies for many illnesses. Hence, their utilization is imperative for the improvement of therapeutic vaccines. In contemporary studies, a plethora of cell-penetrating peptides have been unveiled, each characterized by its own distinct structural attributes and associated mechanisms. Although it is widely acknowledged that there are multiple pathways through which particles might be internalized, a comprehensive understanding of the specific mechanisms by which these particles enter cells has to be fully elucidated. The absorption of cell-penetrating peptides can occur through either direct translocation or endocytosis. However, it is worth noting that categories of cell-penetrating peptides are not commonly linked to specific entrance mechanisms. Furthermore, research has demonstrated that cell-penetrating peptides (CPPs) possess the capacity to enhance antigen uptake by cells and facilitate the traversal of various biological barriers. The primary objective of this work is to examine the mechanisms by which cell-penetrating peptides are internalized by cells and their significance in facilitating the administration of drugs, particularly in the context of gene therapy and vaccine development. The current study investigates the immunostimulatory properties of numerous vaccine components administered using different cell-penetrating peptides (CPPs). This study encompassed a comprehensive discussion on various topics, including the uptake pathways and mechanisms of cell-penetrating peptides (CPPs), the utilization of CPPs as innovative vectors for gene therapy, the role of CPPs in vaccine development, and the potential of CPPs for antigen delivery in the context of vaccine development.

Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

The Regulatory Mechanism of Rab21 in Human Diseases.

Rab proteins are important components of small GTPases and play crucial roles in regulating intracellular transportation and cargo delivery. Maintaining the proper functions of Rab proteins is essential for normal cellular activities such as cell signaling, division, and survival. Due to their vital and irreplaceable role in regulating intracellular vesicle transportation, accumulated researches have shown that the abnormalities of Rab proteins and their effectors are closely related to human diseases. Here, this review focused on Rab21, a member of the Rab family, and introduced the structures and functions of Rab21, as well as the regulatory mechanisms of Rab21 in human diseases, including neurodegenerative diseases, cancer, and inflammation. In summary, we described in detail the role of Rab21 in human diseases and provide insights into the potential of Rab21 as a therapeutic target for diseases.

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids.

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.

Augmenting Intracellular Cargo Delivery of Extracellular Vesicles in Hypoxic Tissues through Inhibiting Hypoxia-Induced Endocytic Recycling.

As mesenchymal stem-cell-derived small extracellular vesicles (MSC-sEVs) have been widely applied in treatment of degenerative diseases, it is essential to improve their cargo delivery efficiency in specific microenvironments of lesions. However, the interaction between the microenvironment of recipient cells and MSC-sEVs remains poorly understood. Herein, we find that the cargo delivery efficiency of MSC-sEVs was significantly reduced under hypoxia in inflammaging nucleus pulposus cells due to activated endocytic recycling of MSC-sEVs. Hypoxia-inducible factor-1 (HIF-1)-induced upregulated RCP (also known as RAB11FIP1) is shown to promote the Rab11a-dependent recycling of internalized MSC-sEVs under hypoxia via enhancing the interaction between Rab11a and MSC-sEV. Based on this finding, si-RCP is loaded into MSC-sEVs using electroporation to overcome the hypoxic microenvironment of intervertebral disks. The engineered MSC-sEVs significantly inhibit the endocytic recycling process and exhibit higher delivery efficiency under hypoxia. In a rat model of intervertebral disk degeneration (IDD), the si-RCP-loaded MSC-sEVs successfully treat IDD with improved regenerative capacity compared with natural MSC-sEV. Collectively, the findings illustrate the intracellular traffic mechanism of MSC-sEVs under hypoxia and demonstrate that the therapeutic capacity of MSC-sEVs can be improved via inhibiting endocytic recycling. This modifying strategy may further facilitate the application of extracellular vesicles in hypoxic tissues.

The Roles of Exosomes in the Diagnose, Development and Therapeutic Resistance of Oral Squamous Cell Carcinoma.

Oral cancer is one of the most common cancers worldwide, of which more than half of patients are diagnosed at a locally advanced stage with poor prognosis due to recurrence, metastasis and resistant to treatment. Thus, it is imperative to further explore the potential mechanism of development and drug resistance of oral cancer. Exosomes are small endosome-derived lipid nanoparticles that are released by cells. Since the cargoes of exosomes were inherited from their donor cells, the cargo profiles of exosomes can well recapitulate that of their donor cells. This is the theoretical basis of exosome-based liquid biopsy, providing a tool for early diagnosis of oral cancer. As an important intracellular bioactive cargo delivery vector, exosomes play a critical role in the development of oral cancer by transferring their cargoes to receipt cells. More importantly, recent studies have revealed that exosomes could induce therapy-resistance in oral cancer through multiple ways, including exosome-mediated drug efflux. In this review, we summarize and compare the role of exosomes in the diagnosis, development and therapy-resistant of oral cancer. We also highlight the clinical application of exosomes, and discuss the advantages and challenges of exosomes serving as predictive biomarker, therapy target and therapy vector in oral cancer.

Design and fabrication of intracellular therapeutic cargo delivery systems based on nanomaterials: current status and future perspectives.

Intracellular cargo delivery, the introduction of small molecules, proteins, and nucleic acids into a specific targeted site in a biological system, is an important strategy for deciphering cell function, directing cell fate, and reprogramming cell behavior. With the advancement of nanotechnology, many researchers use nanoparticles (NPs) to break through biological barriers to achieving efficient targeted delivery in biological systems, bringing a new way to realize efficient targeted drug delivery in biological systems. With a similar size to many biomolecules, NPs possess excellent physical and chemical properties and a certain targeting ability after functional modification on the surface of NPs. Currently, intracellular cargo delivery based on NPs has emerged as an important strategy for genome editing regimens and cell therapy. Although researchers can successfully deliver NPs into biological systems, many of them are delivered very inefficiently and are not specifically targeted. Hence, the development of efficient, target-capable, and safe nanoscale drug delivery systems to deliver therapeutic substances to cells or organs is a major challenge today. In this review, on the basis of describing the research overview and classification of NPs, we focused on the current research status of intracellular cargo delivery based on NPs in biological systems, and discuss the current problems and challenges in the delivery process of NPs in biological systems.

Breaking free: endocytosis and endosomal escape of extracellular vesicles

Extracellular vesicles (EVs) are natural micro-/nanoparticles that play an important role in intercellular communication. They are secreted by producer/donor cells and subsequent uptake by recipient/acceptor cells may result in phenotypic changes in these cells due to the delivery of cargo molecules, including lipids, RNA, and proteins. The process of endocytosis is widely described as the main mechanism responsible for cellular uptake of EVs, with endosomal escape of cargo molecules being a necessity for the functional delivery of EV cargo. Equivalent to synthetic micro-/nanoparticles, the properties of EVs, such as size and composition, together with environmental factors such as temperature, pH, and extracellular fluid composition, codetermine the interactions of EVs with cells, from binding to uptake, intracellular trafficking, and cargo release. Innovative assays for detection and quantification of the different steps in the EV formation and EV-mediated cargo delivery process have provided valuable insight into the biogenesis and cellular processing of EVs and their cargo, revealing the occurrence of EV recycling and degradation, next to functional cargo delivery, with the back fusion of the EV with the endosomal membrane standing out as a common cargo release pathway. In view of the significant potential for developing EVs as drug delivery systems, this review discusses the interaction of EVs with biological membranes en route to cargo delivery, highlighting the reported techniques for studying EV internalization and intracellular trafficking, EV-membrane fusion, endosomal permeabilization, and cargo delivery, including functional delivery of RNA cargo.

Abstract P1048: Dual Myristic Acid And Trans Activator Of Transcription Conjugated Peptide Facilitates Delivery Of Protein Kinase C Beta II Peptide Inhibitor To Attenuate Myocardial Ischemia/Reperfusion Injury

Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 μM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration. The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min) . Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) from base to apex and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis. Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p&lt;0.05), 1 nM (10.0±2.3%, n=5, p&lt;0.05), to 100 pM (12.6±2.3%, n=5, p&lt;0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p&lt;0.05). The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts to restore post-reperfusion cardiac function combined with significant cardiac salvaging effect (reduced infarct size) in a porcine myocardial I/R model.

Recent Advances in Microscale Electroporation.

Electroporation (EP) is a commonly used strategy to increase cell permeability for intracellular cargo delivery or irreversible cell membrane disruption using electric fields. In recent years, EP performance has been improved by shrinking electrodes and device structures to the microscale. Integration with microfluidics has led to the design of devices performing static EP, where cells are fixed in a defined region, or continuous EP, where cells constantly pass through the device. Each device type performs superior to conventional, macroscale EP devices while providing additional advantages in precision manipulation (static EP) and increased throughput (continuous EP). Microscale EP is gentle on cells and has enabled more sensitive assaying of cells with novel applications. In this Review, we present the physical principles of microscale EP devices and examine design trends in recent years. In addition, we discuss the use of reversible and irreversible EP in the development of therapeutics and analysis of intracellular contents, among other noteworthy applications. This Review aims to inform and encourage scientists and engineers to expand the use of efficient and versatile microscale EP technologies.