Intracellular Delivery Research Articles

ADVANCING MESSENGER RNA THERAPEUTICS FOR ORTHO-REGENERATION

Messenger RNA (mRNA) is a new class of drug that can be used to express a therapeutic protein and, in contrast to DNA, is safer and inexpensive. Among its advantages, mRNA will immediately begin to express its encoded protein in the cell cytoplasm. The protein will be expressed for a period of time, after which the mRNA is degraded. There is no risk of genetic damage, one of the concerns with plasmid DNA (pDNA) used in traditional gene therapy approaches. Nevertheless, mRNA application in tissue regeneration and regenerative medicine remains limited. In this case, mRNA must overcome its main hurdles: immunogenicity, lack of stability, and intracellular delivery. Research has been done to overcome these limitations, and the future of mRNA seems promising for tissue repair1,2. This keynote talk will address questions including: What are the opportunities for mRNA to improve outcomes in musculoskeletal tissue repair, in particular bone and cartilage? What are the key factors and challenges to expediting this technology to patient treatment (beyond COVID-19 vaccination)?Acknowledgements: E.R.B thanks the cmRNAbone project funded by the European Union's Horizon 2020 research and innovation program under the grant agreement no. 874790 and the NIH R01 AR074395 from NIAMS for funding her mRNA work.

High throughput intracellular delivery by viscoelastic mechanoporation

Brief pulses of electric field (electroporation) and/or tensile stress (mechanoporation) have been used to reversibly permeabilize the plasma membrane of mammalian cells and deliver materials to the cytosol. However, electroporation can be harmful to cells, while efficient mechanoporation strategies have not been scalable due to the use of narrow constrictions or needles which are susceptible to clogging. Here we report a high throughput approach to mechanoporation in which the plasma membrane is stretched and reversibly permeabilized by viscoelastic fluid forces within a microfluidic chip without surface contact. Biomolecules are delivered directly to the cytosol within seconds at a throughput exceeding 250 million cells per minute. Viscoelastic mechanoporation is compatible with a variety of biomolecules including proteins, RNA, and CRISPR-Cas9 ribonucleoprotein complexes, as well as a range of cell types including HEK293T cells and primary T cells. Altogether, viscoelastic mechanoporation appears feasible for contact-free permeabilization and delivery of biomolecules to mammalian cells ex vivo.

Light-controllable cell-membrane disturbance for intracellular delivery.

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.

"Yin-Yang philosophy" for the design of anticancer drug delivery nanoparticles.

Understanding the in vivo transport process provides guidelines for designing ideal nanoparticles (NPs) with higher efficacy and fewer off-target effects. Many factors, such as particle size, morphology, surface potential, structural stability, and etc., may influence the delivering process of NPs due to the existence of various physiological barriers within the body. Herein, we summarise the distinct influences of NP physicochemical properties on the four consecutive in vivo transport steps: (1) navigating with bloodstream within blood vessels, (2) transport across vasculature walls into tumour tissues, (3) intratumoural transport through the interstitial space, and (4) cellular uptake & intracellular delivery by cancerous cells. We found that the philosophy behind the current consensus for NP design has certain similarities to the "Yin-Yang" theory in traditional Chinese culture. Almost all physicochemical properties, regardless of big or small sizes, long or short length, positive or negative zeta potentials, are double-edged swords. The balance of potential benefits and side effects, drug selectivity and accessibility should be fully considered when optimising particle design, similar to the "Yin-Yang harmony". This paper presents a comprehensive review of the advancements in NPs research, focusing on their distinct features in tumour targeting, drug delivery, and cell uptake. Additionally, it deliberates on future developmental trends and potential obstacles, thereby aiming to uncover the ways these characteristics influence the NPs' biological activity and provide theoretical guidance for the targeted delivery of NPs.

A bioinspired pseudopeptide-based intracellular delivery platform enhances the cytotoxicity of a ribosome-inactivating protein through multiple death pathways.

Saporin is a 28 621 Da protein and plant toxin possessing rRNA N-glycosidase activity. Due to its potent ribosome-inactivating ability, saporin is commonly studied as an anticancer agent. However, its enzymatic activity is greatly hindered by its poor plasma membrane permeability. To overcome this barrier, we used a bioinspired intracellular delivery platform based on the pH-responsive pseudopeptide, poly(L-lysine isophthalamide) grafted with L-phenylalanine at a stoichiometric molar percentage of 50% (PP50). PP50 was co-incubated with saporin (PP50/saporin) in a mildly acidic pH environment to aid intracellular delivery and increase saporin's therapeutic potential. We demonstrated that PP50 greatly enhanced the cytotoxicity of saporin in the 2D monolayer of A549 cells and 3D A549 multicellular spheroids whilst remaining non-toxic when administered alone. To elucidate the mechanism of cell death, we assessed the activation of caspases, the inhibition of protein synthesis, the onset of apoptosis and the mechanism of PP50/saporin entry. Inhibition of protein synthesis and activation of caspases 3/7, 8 and 9 were found to occur before the onset of apoptosis and cell death. PP50/saporin was also shown to rely on micropinocytosis and caveolae-mediated endocytosis for cell entry. In addition, fluorescein isothiocyanate-labelled saporin (FITC-saporin) was localized within the cytoplasm and nuclei when delivered with Cyanine5-labelled PP50 (Cy5-PP50). Taken together, this suggests that multiple pathways are triggered to initiate apoptosis and cell death in cells treated with PP50/saporin. Therefore, these results make PP50 a potential intracellular delivery platform for the internalization of protein therapeutics.

Ultra-low intensity light pulses for large cargo delivery into hard-to-transfect cells using an rGO mixed PDMS microtip device.

Nanoparticle-mediated photoporation has arisen as a universal intracellular delivery tool; however, the direct interaction of nanoparticles and cells hampers its clinical translation. Here, we report a uniform contactless intracellular delivery that transfects a large number of cells within a minute and avoids direct contact of nanoparticles and cells, thereby improving the cell viability. Our platform consists of an array of polydimethylsiloxane (PDMS) mixed reduced graphene oxide (rGO) nanoflakes on pyramidal microtips, uniformly distributed at the apex of the tip. The extraordinary optoelectronic properties of rGO were combined with micro-pyramidal cavities to entrap light in micro-cavities and efficiently convert it into heat through multiple reflections and absorptions. As a result, ultralow infra-red laser pulse irradiation could create cavitation bubbles followed by cell membrane deformation and biomolecular delivery. Using this delivery platform, we have achieved the delivery of small to large cargo (668 Da to 465 kDa) in various mammalian cells, including hard-to-transfect H9C2 cardiomyocytes. The best results were achieved for enzyme (465 kDa) delivery with a transfection efficiency and cell viability of 95% and 98%, respectively, in SiHa cells. The highly efficient cargo delivery tool demonstrated a safe and effective approach for cell therapy and diagnostics.

A computational study of cell membrane damage and intracellular delivery in a cross-slot microchannel.

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.

Flash nanoprecipitation assisted self-assembly of ionizable lipid nanoparticles for nucleic acid delivery.

Ionizable lipid nanoparticles (LNPs) have emerged as a powerful tool for the intracellular delivery of nucleic acids. Following the recent success of LNP-based siRNA therapeutics and mRNA vaccines, the use of ionizable lipids for nucleic acid delivery has tremendously increased. Here, we introduce a flash nanoprecipitation (FNP) approach using the confined impingement (CIJ) mixer to stably self-assemble ionizable LNPs. To validate this approach, we employed three clinically relevant LNP formulations containing SM102, ALC0315, and DLin-MC3-DMA as ionizable lipids. FNP-assembled LNPs showed >95% encapsulation efficiency of mRNA and siRNA payloads and particle sizes below 150 nm. SM102 or ALC0315 LNPs demonstrated efficient delivery of mRNA into immune cells in vitro and to lymphoid organs in vivo, whereas Dlin-MC3-DMA LNPs allowed effective intracellular siRNA delivery with great functional ability. The FNP technique could economically produce LNPs in smaller volumes that are highly suitable for the discovery phase.

Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

Intracellular delivery, imaging and drug-sensing using a plasmonic-enhanced hybrid nanostystem

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.

Cationic dextrin nanoparticles for effective intracellular delivery of cytochrome C in cancer therapy

Intracellular protein delivery using cationic dextrin nanoparticles show promise as a selective and specific approach to cancer therapy.

Intracellular delivery and deep tissue penetration of nucleoside triphosphates using photocleavable covalently bound dendritic polycations.

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.

Intracellular Protein Delivery: Approaches, Challenges, and Clinical Applications.

Protein biologics are powerful therapeutic agents with diverse inhibitory and enzymatic functions. However, their clinical use has been limited to extracellular applications due to their inability to cross plasma membranes. Overcoming this physiological barrier would unlock the potential of protein drugs for the treatment of many intractable diseases. In this review, we highlight progress made toward achieving cytosolic delivery of recombinant proteins. We start by first considering intracellular protein delivery as a drug modality compared to existing Food and Drug Administration-approved drug modalities. Then, we summarize strategies that have been reported to achieve protein internalization. These techniques can be broadly classified into 3 categories: physical methods, direct protein engineering, and nanocarrier-mediated delivery. Finally, we highlight existing challenges for cytosolic protein delivery and offer an outlook for future advances.

A self-assembled copper-artemisinin nanoprodrug as an efficient reactive oxygen species amplified cascade system for cancer treatment.

Chemodynamic therapy (CDT) is a tumor-specific intervention methodology, which is based on the upregulation of reactive oxygen species (ROS) content by triggering the Fenton or Fenton-like reaction within the tumor microenvironment (TME). However, there are still challenges in achieving high-efficiency CDT on account of both the limited intracellular hydrogen peroxide (H2O2) and delivery efficiency of Fenton metal ions. Copper-based nanotherapeutic systems have attracted extensive attention and have been widely applied in the construction of nanotherapeutic systems and multimodal synergistic therapy. Herein, we propose a strategy to synergize chemotherapy drugs that upregulate intracellular ROS content with chemodynamic therapy and construct an artemisinin-copper nanoprodrug for proof-of-concept. With the proposed biomimetic self-assembly strategy, we successfully construct an injectable nanoprodrug with suitable size distribution and high drug loading content (68.1 wt%) through the self-assembly of amphiphilic artemisinin prodrug and copper ions. After reaching the TME, both Cu2+ ions and free AH drugs can be released from AHCu nanoprodrugs. Subsequently, the disassembled Cu2+ ions are converted into Cu+ ions by consuming the intracellular GSH. The generated Cu+ ions serve as a highly efficient Fenton-like reagent for robust ROS generation from both AH and tumor-over-produced H2O2. Results show that the nanoprodrug can realize the cascade amplification of ROS generation via artemisinin delivery and subsequent in situ Fenton-like reaction and a high tumor inhibition rate of 62.48% in vivo. This work provides a promising strategy for the design and development of an efficient nanoprodrug for tumor-specific treatment.

Cytosolic delivery of cytochrome c conjugates induces apoptosis at nanomolar levels through a caspase-3-dependent pathway.

Cytochrome c (CytC) is conjugated with a small molecule TG6 to give TG6-CytC, which is directly delivered into cytosol, triggering the release of endogenous CytC from mitochondria, and inducing a caspase-3-dependent apoptosis with an IC50 down to 2.4 nM. This work shows an efficient strategy for intracellular protein delivery.

Ion Doped Hollow Silica Nanoparticles as Promising Oligonucleotide Delivery Systems to Mesenchymal Stem Cells.

Oligonucleotide (ON) therapy is a promising treatment for a wide range of complex genetic disorders, but inefficient intracellular ON delivery has hindered clinical translation. Hollow silica nanoparticles (HSN) hold potential as effective ON delivery vehicles since ON can be encapsulated in the hollow core in situ where they are protected from degradation by eg nucleases. However, HSN must be modified to allow degradation and subsequent (sub)cellular ON release. In this report, we investigated the use of ion and fluorescent dye co-doping in the HSN silica matrix to enable HSN degradability and in vitro visualization. HSN were core encapsulated with ON, doped with Ca2+, Cu2+, Zn2+, Se2+ and Sr2+ ions and co-condensed with rhodamine b isothiocyanate (RITC) by a modified reverse microemulsion method. HSN were physiochemically characterized and their biological activity such as uptake and toxicity were evaluated in mesenchymal stem cells (hMSCs). We successfully doped HSN with RITC and Ca2+, Cu2+, Zn2+ and Sr2+ ions. We observed that doping HSN with Ca2+ and Sr2+ enhanced RITC incorporation while ON encapsulation in HSN increased Cu2+ and Zn2+ doping efficiency. Moreover, our dual-doped HSN demonstrated controlled ON release in the presence of intracellular mimicking levels of glutathione (GSH) and limited release in the absence of GSH over 14 days. HSN were biocompatible in hMSCs up to 300µg/mL except for Cu2+ doped HSNs which were cytotoxic even at ~10µg/mL. HSN uptake was influenced by the dopant ion, DNA encapsulation, and HSN concentration, where Zn-HSN showed the lowest and Sr-HSN and Se-HSND, the highest uptake in hMSCs. We report a straightforward one-pot procedure to create ion and fluorescent dye co-doped HSN that can efficiently incorporate ON, as promising new gene vectors.

A novel nanocomposite drug delivery system for SARS-CoV-2 infections

To develop an inhalable drug delivery system, we synthesized Remdesivir-loaded nanocomposites (RDV NCs) by coating drug-loaded nanoparticles with supramolecular cell-penetrating peptide nanofibers, enhancing uptake and intracellular drug delivery.

Bortezomib-encapsulated metal-phenolic nanoparticles for intracellular drug delivery.

Bortezomib (BTZ) is an important boronate proteasome inhibitor that is widely used in cancer therapy. However, the clinical application of BTZ suffers from poor stability and serious adverse effects. Herein, we fabricated metal-polyphenol nanoparticles for the covalent encapsulation of BTZ. BTZ-encapsulated tannic acid (TA)-Fe3+ nanoparticles can be prepared by mixing BTZ, TA, and ferric chloride owing to the formation of metal-polyphenol coordination interaction and dynamic boronate ester bonds. The BTZ-encapsulated TA-Fe3+ nanoparticles (BTZ NPs) are stable in physiological environment (pH 7.4) with minimal drug leakage. However, BTZ NPs can be disassembled in an acidic environment. Therefore, BTZ can be rapidly released from BTZ NPs in an acidic environment (pH 5.0). More than 50% BTZ can be released from BTZ NPs after 8 h incubation at pH 5.0. BTZ NPs exhibited high cytotoxicity against human osteosarcoma Saos-2 cells and human multiple myeloma OPM-2 cells. The metal-polyphenol nanoparticles can be a promising nanoplatform for the delivery of BTZ with simultaneously enhanced therapeutic efficacy and reduced side effects.

Furan-rich, biobased transfection agents as potential oligomeric candidates for intracellular plasmid DNA delivery.

Biobased, DNA delivery vectors have been synthesized with a core motif composed of 2,5-bishydroxymethylfuran (BHMF) readily available from an important biomass feedstock 5-hydroxymethyl furfural (HMF). To generate the product, BHMF was first converted to 2,5-furan bishydroxymethyl diacrylate (2,5-FDA), which was later conjugated with different types of secondary amines. Rich in tertiary nitrogen, these oligomeric FDA-amino esters demonstrated stable electrostatic interactions with negatively charged plasmid DNA in an aqueous environment. We evaluated synthetic routes toward these plasmid DNA-binding amino esters (pFASTs), identified their nanoscale features, and attempted to establish their structure-property relationship in the context of the DNA delivery. Our preliminary studies show that the pFASTs formed stable complexes with the plasmid DNA. Dynamic light scattering indicated that the DNA polyplexes of pFASTs have hydrodynamic diameters within the size range of 100-150 nm with a surface charge (ζ-potential) ranging from -10 to +33 mV, depending on pFAST type. These oligomeric amino esters rich in furan motif were also found to successfully transfect the GFP-expressing plasmid DNA intracellularly. Collectively, this study establishes a new route to produce DNA transfection agents from sustainable resources that can be used for transferring genetic materials for humans, veterinary, and agrochemical purposes.

Albumin nanocapsules and nanocrystals for efficient intracellular drug release.

In order to achieve a therapeutic effect, many drugs have to reach specific cellular compartments. Nanoscale drug delivery systems extend the circulation time, reduce adverse effects and thus improve tolerability compared to systemic administration. We have developed two types of albumin-coated nanocarriers equipped with built-in dyes to track their cellular uptake and intracellular enzymatic opening. Using the approved antiprotozoal drug and STAT3 inhibitor Atovaquone (Ato) as prototype for a hydrophobic small molecule, we show that Ato-loaded ovalbumin-coated nanocapsules (Ato-nCap) preferentially enter human myeloid cells. In contrast, Ato nanocrystals coated with human serum albumin (Ato-nCry) distribute their cargo in all different immune cell types, including T and B cells. By measuring the effect of Ato nanocarriers on induced STAT3 phosphorylation in IL-10-primed human dendritic cells and constitutive STAT3 phosphorylation in human melanoma cells, we demonstrate that the intracellular Ato release is particularly effective from Ato nanocrystals and less toxic than equal doses of free drug. These new nanocarriers thus represent effective systems for intracellular drug delivery.