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Protein drugs are a critically important therapeutic modality due to the sophisticated binding recognition, catalytic properties, and disease relevance of proteins. There is a clear need for new strategies able to improve pharmacokinetics, bioavailability, and/or intracellular delivery of therapeutic proteins, as stability limitations have significantly hindered clinical advancement, and most proteins are membrane impermeable. Bioconjugation strategies able to site-specifically modify proteins with cell binding, and other ligands offer a particularly valuable approach to facilitate protein delivery due to the importance of ligand presentation on protein bioactivity and cellular uptake. We explored unnatural amino acid (UAA) incorporation as a novel strategy to tunably incorporate clustered cell-binding ligands in fluorescent proteins and suicide enzymes, resulting in substantial increases in cell-specific uptake and targeted cell-killing activity. These approaches offer a valuable and versatile method to modify a variety of proteins and enable improved clinical potential., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3β[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pK a values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency., (© 2024 Wiley‐VCH GmbH.)

We propose a three-dimensional computational framework to simulate the flow-induced cell membrane damage and the resulting enhanced intracellular mass transport in a cross-slot microchannel. We model the cell as a liquid droplet enclosed by a viscoelastic membrane and solve the cell deformation using a well-tested immersed-boundary lattice-Boltzmann method. The cell membrane damage, which is directly related to the membrane permeability, is considered using continuum damage mechanics. The transport of the diffusive solute into the cell is solved by a lattice-Boltzmann model. After validating the computational framework against several benchmark cases, we consider a cell flowing through a cross-slot microchannel, focusing on the effects of the flow strength, channel fluid viscosity and cell membrane viscosity on the membrane damage and enhanced intracellular transport. Interestingly, we find that under a comparable pressure drop across the device, for cells with low membrane viscosity, the inertial flow regime, which can be achieved by driving a low-viscosity liquid at a high speed, often leads to much larger membrane damage, compared with the high-viscosity low-speed viscous flow regime. However, the enhancement can be significantly reduced or even reversed by an increase of the cell membrane viscosity, which limits cell deformation, particularly in the inertial flow regime. Our computational framework and simulation results may guide the design and optimisation of microfluidic devices, which use cross-slot geometry to disrupt cell membranes to enhance intracellular delivery of solutes.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

Nucleoside triphosphates (NTPs) are essential in various biological processes. Cellular or even organismal controlled delivery of NTPs would be highly desirable, yet in cellulo and in vivo applications are hampered owing to their negative charge leading to cell impermeability. NTP transporters or NTP prodrugs have been developed, but a spatial and temporal control of the release of the investigated molecules remains challenging with these strategies. Herein, we describe a general approach to enable intracellular delivery of NTPs using covalently bound dendritic polycations, which are derived from PAMAM dendrons and their guanidinium derivatives. By design, these modifications are fully removable through attachment on a photocage, ready to deliver the native NTP upon irradiation enabling spatiotemporal control over nucleotide release. We study the intracellular distribution of the compounds depending on the linker and dendron generation as well as side chain modifications. Importantly, as the polycation is bound covalently, these molecules can also penetrate deeply into the tissue of living organisms, such as zebrafish., Competing Interests: The authors declare no conlicts of interest., (This journal is © The Royal Society of Chemistry.)

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

A hallmark of the last decades is an extensive development of genome editing systems and technologies propelling genetic engineering to the next level. Specific and efficient delivery of genome editing tools to target cells is one of the key elements of such technologies. Conventional vectors are not always suitable for this purpose due to a limited cargo volume, risks related to cancer and immune reactions, toxicity, a need for high-purity viral material and quality control, as well as a possibility of integration of the virus into the host genome leading to overexpression of the vector components and safety problems. Therefore, the search for novel approaches to delivering proteins and nucleic acids into cells is a relevant priority. This work reviews abiotic vectors and systems for delivering genome editing tools into target cells, including liposomes and solid lipid particles, other membrane-based vesicles, cell-penetrating peptides, micelles, dendrimers, carbon nanotubes, inorganic, polymer, metal and other nanoparticles. It considers advantages, drawbacks and preferred applications of such systems as well as suitability thereof for the delivery of genome editing systems. A particular emphasis is placed on metal-organic frameworks (MOFs) and their potential in the targeted intracellular delivery of proteins and polynucleotides. It has been concluded that further development of MOF-based vectors and technologies, as well as combining MOFs with other carriers can result in safe and efficient delivery systems, which would be able to circulate in the body for a long time while recognizing target cells and ensuring cell-specific delivery and release of intact cargoes and, thereby, improving the genome editing outcome., Competing Interests: The authors declare no conflict of interest., (Copyright © AUTHORS.)

The success of personalized medicine in oncology relies on using highly effective and precise therapeutic modalities such as small interfering RNA (siRNA) and monoclonal antibodies (mAbs). Unfortunately, the clinical exploitation of these biological drugs has encountered obstacles in overcoming intricate biological barriers. Drug delivery technologies represent a plausible strategy to overcome such barriers, ultimately facilitating the access to intracellular domains. Here, an overview of the current landscape on how nanotechnology has dealt with protein corona phenomena as a first and determinant biological barrier is presented. This continues with the analysis of strategies facilitating access to the tumor, along with conceivable methods for enhanced tumor penetration. As a final step, the cellular barriers that nanocarriers must confront in order for their biological cargo to reach their target are deeply analyzed. This review concludes with a critical analysis and future perspectives of the translational advances in personalized oncological nanomedicine., (© 2023 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Summary: Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.

Abstract Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor‐T (CAR‐T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are outlined.

Metastasis stands as the leading cause of mortality among colorectal cancer (CRC) patients. Galunisertib (LY2157299, LY) is a small molecule demonstrating promising anti-cancer effects by targeting the Transforming Growth Factor-beta (TGF-β) pathway. This route plays a pivotal role in initiating the epithelial-to-mesenchymal transition (EMT), a critical process for metastatic spread. Unfortunately, LY chronic treatment causes undesired effects. To mitigate these side effects, nanoscale drug delivery systems have emerged as a transformative approach in cancer treatment, enhancing drug effectiveness while minimizing toxicity. In this study, we introduce a hybrid nanosystem (DNP-AuNPs-LY@Gel) comprising porous diatomite nanoparticles decorated with plasmonic gold nanoparticles (AuNPs), encapsulating LY within a gelatin shell. This multifunctional nanosystem demonstrates efficient LY delivery, EMT reversal in CRC 2D and 3D cultures, and anti-cancer effects in vivo. Moreover, the nanosystem allowed the quantification with sub-femtogram resolution of the drug intracellularly released using surface-enhanced Raman spectroscopy (SERS). The release of LY is triggered by CRC cell acidic microenviroment. Real-time monitoring of drug release at the single-cell level is achieved by analyzing SERS signals of LY within CRC cells. The heightened efficacy of LY delivery through the DNP-AuNPs-LY@Gel complex offers a promising alternative strategy for reducing drug dosages and subsequent undesired effects.

Virus-like particles (VLPs) are nanostructures assemble from viral proteins. Besides widely used for vaccine development, VLPs have also been explored as nanocarriers for cargo delivery as they combine the key advantages of viral and non-viral vectors. While it protects cargo molecules from degradation, the VLP has good cell penetrating property to mediate cargo passing the cell membrane and released into cells, making the VLP an ideal tool for intracellular delivery of biomolecules and drugs. Great progresses have been achieved and multiple challenges are still on the way for broad applications of VLP as delivery vectors. Here we summarize current advances and applications in VLP as a delivery vector. Progresses on delivery of different types of biomolecules as well as drugs by VLPs are introduced, and the strategies for cargo packaging are highlighted which is one of the key steps for VLP mediated intracellular delivery. Production and applications of VLPs are also briefly reviewed, with a discussion on future challenges in this rapidly developing field.

This study explores the potential of laser-induced nano-photon-poration as a non-invasive technique for the intracellular delivery of micro/macromolecules at the single-cell level. This research proposes the utilization of gold-coated spiky polymeric nanoparticles (Au-PNPs) and gold nanorods (GNRs) to achieve efficient intracellular micro/macromolecule delivery at the single-cell level. By shifting the operating wavelength towards the near-infrared (NIR) range, the intracellular delivery efficiency and viability of Au-PNP-mediated photon-poration are compared to those using GNR-mediated intracellular delivery. Employing Au-PNPs as mediators in conjunction with nanosecond-pulsed lasers, a highly efficient intracellular delivery, while preserving high cell viability, is demonstrated. Laser pulses directed at Au-PNPs generate over a hundred hot spots per particle through plasmon resonance, facilitating the formation of photothermal vapor nanobubbles (PVNBs). These PVNBs create transient pores, enabling the gentle transfer of cargo from the extracellular to the intracellular milieu, without inducing deleterious effects in the cells. The optimization of wavelengths in the NIR region, coupled with low laser fluence (27 mJ/cm 2 ) and nanoparticle concentrations (34 µg/mL), achieves outstanding delivery efficiencies (96%) and maintains high cell viability (up to 99%) across the various cell types, including cancer and neuronal cells. Importantly, sustained high cell viability (90-95%) is observed even 48 h post laser exposure. This innovative development holds considerable promise for diverse applications, encompassing drug delivery, gene therapy, and regenerative medicine. This study underscores the efficiency and versatility of the proposed technique, positioning it as a valuable tool for advancing intracellular delivery strategies in biomedical applications.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are promising candidates for disease modeling and therapeutic purposes, however, non-viral intracellular delivery in these cells remains challenging. Gold nanoparticle (AuNP)-sensitized photoporation creates transient pores in the cell membrane by vapor nanobubble formation, allowing diffusion of extracellular biomolecules. This non-viral technique was employed to test and optimize its distinct physical mode of action in iPSC-CMs. Photoporation optimization was aimed at achieving high delivery rates while minimizing cell death. Various AuNP concentrations, in conjunction with different laser fluences, were explored to facilitate the intracellular delivery of 10 kDa and 150 kDa FITC-labelled dextran as model macromolecules. Cardiomyocyte viability was assessed using the CellTiter-Glo® viability assay, while the delivery efficiency was quantified through flow cytometry. On 30 day-old cardiomyocytes, AuNP photoporation was able to yield ∼60 % delivery efficiency while maintaining a high cell viability (∼70 %). Overall, higher AuNP concentrations resulted in greater delivery efficiencies, albeit at the expense of lower cell viability. Finally, photoporation was capable of patterning a geometric shape, demonstrating its exceptional selective resolution in delivering molecules to spatially restricted regions of the cell culture. In conclusion, AuNP-photoporation exhibits considerable potential as an effective and gentle non-viral method for intracellular delivery in iPSC-CMs.•AuNP-photoporation is a non-viral intracellular delivery method suitable for iPSC-CMs with high efficiency and cell viability•This method is capable of spatially resolved intracellular delivery with excellent resolution., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KB declares financial interest in the company Trince which markets the LumiPore photoporation technology, (© 2024 The Authors. Published by Elsevier B.V.)