Abstract
Wound Healing
Ceria Nanocrystals Decorated Mesoporous Silica Nanoparticle-Based ROS-Scavenging Tissue Adhesive for Highly Efficient Regenerative Wound Healing
The goal of wound healing is the restoration of tissue integrity and function, while minimizing aesthetic impact following injury. Currently, while several options, including staples, sutures, and tissue adhesives, exist, perfect wound regeneration rather than fibrotic healing is still an issue. An emerging approach is to focus on host intrinsic regenerative capacity and leverage it for active wound regeneration. Following skin injury, reactive oxygen species (ROS) is generated in large amounts in the wound microenvironment as a defense mechanism against pathogens. While pathogen defense is favorable, wound repair may be hampered by excessive quantities of ROS. It potentially damages biomolecules, including proteins, DNA, lipids, and carbohydrates. Excessive quantities even give rise to such consequences as cellular senescence, fibrotic scarring, and inflammation. Thus, an appropriate balance of ROS is required to balance the requirements of warding off infections and skin restoration. A role of reduced ROS was further evident during the regeneration and recovery of injury in mice heart tissue. Thus, fabricating suitable biomaterials to manage ROS levels and suppress oxidative damage is one strategy to promote regenerative wound healing.
Nanostructure biomaterials are interesting because they can mimic extracellular components on a nanoscale to enable interactions between materials and biological entities. Ceria nanocrystals have been demonstrated to reduce ROS, thus protecting cells against oxidative stress due to a combination of reduced and oxidized ion species on its surface that reversibly bind to oxygen. These have been found to be effective against such diseases as ischemic stroke and autoimmune degenerative diseases.
The ceria nanocrystals were then immobilized on mesoporous silica nanoparticles (MSNs). MSNs have emerged as a promising nanomaterial to achieve efficient wound healing due to nanobridging between materials and biological tissue matrix.
Using reverse micelle fabrication, the MSN surface was functionalized with amine groups to immobilize ceria nanocrystals. Equipment, such as scanning transmission electron microscopy (STEM) and energy-dispersive x-ray spectroscopy (EDX), confirm that nanocrystals were distributed on the surface. Following functionalization, the particle size increased and the zeta potential decreased. Evaluation of the MSN-ceria was performed by attaching two pieces of rat skin. This displayed similar maximum adhesive strength with or without ceria modification, showing that it negligibly influenced tissue adhesion. Efficient nanobridging was also observed in cutaneous wound models. When the ceria nanocrystals were suspended in an aqueous suspension, severe aggregation impeded ROS-scavenging activity. On the other hand, well-dispersed MSN-ceria exhibited good ROS-scavenging activity.
MSN-ceria was further found to have good biocompatibility through cell viability and morphology evaluations. Upon treatment with cells, the intensity of 2′,7′-dichlorofluorescein (DCF) fluorescence (an intracellular ROS assay) of the MSN-ceria pretreated group was significantly diminished. Its pretreatment also resulted in decreased SA-β-gal (senescence) activity.
Using Sprague Dawley (SD) rats to evaluate in vivo wound healing, 2 cm incisions were generated and treated with MSN (without ceria) or MSN-ceria. MSN-ceria-treated wounds gave rise to significantly lower ROS signals and a lower local inflammatory response at the wound site. This demonstrated that MSN-ceria treatment could suppress oxidative and inflammatory responses in animal subjects. MSN-ceria treatment also gave rise to native collagen deposition and alignment like unwounded skin, whereas MSN and vehicle controls had lower collagen deposition. This demonstrated how MSN-ceria effectively gave rise to wound healing without significant scar formation.
Furthermore, MSN-ceria promoted greater skin morphogenesis, including higher expression of sebaceous gland markers (stearoyl-CoA desaturase 1), hair follicle stem and progenitor cell markers, and placenta-expressed transcript-1. In addition, platelet-derived growth factor-a (PDGF-a), responsible for cell proliferation, migration, and division, was also accelerated. Thus, MSN-ceria treatment not only quickens wound closure but also promotes the generation of high-quality healed skin.
Overall, the nanomaterial MSN-ceria was found to have good adhesion strength, making it suitable to bridge wounds. It is able to generate ROS-scavenging ability, which accelerates cutaneous wound healing to promote wound tissue regeneration. The authors see the MSN-ceria strategy as potentially applicable to other wound repair/regeneration sensitive to ROS and tissue adhesion. (Wu, H.; et al. Biomaterials
In Vivo Reprogramming of Wound-Resident Cells Generates Skin Epithelial Tissue
Wounds require keratinocyte (outer layer of skin cells) migration from the adjacent areas to promote re-epithelialization. For large wounds, this process is inefficient, since the epidermis maintains homeostasis (protecting against environmental insults) and prevents water loss. The authors developed an approach involving cellular reprogramming to generate new cells to re-epithelialize the surface of a cutaneous ulcer.
A comparison of primary keratinocytes and human dermal fibroblasts (hDFs) identified 55 transcription factors and 31 microRNAs potentially involved in keratinocyte maturation. A combination of 28 transcription factors induced through lentiviruses led to the development of keratinocyte-like cells in vitro (through keratin, KRT14, and cadherin 1 expression) that generated stratified epithelium in 3D culture using hDFs and adipose-derived stromal cells (hADSCs). These were termed 28TF-induced stratified epithelial progenitors (28TF-iSEPs). Further optimization steps revealed that the optimal combination of reprogramming factors was DNP63A, GRHL2, TFAP2A, and MYC (DGTM factors).
To evaluate the efficiency of the DGTM factors in vivo, an assay was developed by isolating skin ulcers from large cutaneous ulcers. The ulcer constructs were tagged with genetic reporters that generate fluorescence in cells that express Krt(keratin)14. The DGTM factors gave rise to epithelial tissue; this was similarly repeated for mesenchymal cells. Among the different reprogramming factors, all DNP63A factors gave rise to highly clonogenic epithelial cells. Multiple wounding of the reprogrammed skin demonstrated that it was functional, successfully connecting to the surrounding epidermis while retaining its stratified epithelium. Histology analysis over a 90- to 110-day period showed that DGTM-generated skin epithelium had similar characteristics to normal skin.
A further aspect of functional performance involved barrier penetration studies using toluidine blue and lucifer yellow dye. Its transepidermal water loss (TEWL) exhibited equivalent values to intact skin.
This DGTM strategy was then implemented clinically on (7 days) older ulcers by administering DGTM adenoviruses (adeno-associated viruses [AAVs]) through collagen gels. Applying the bioactive molecules—FGF2 and a Rock inhibitor—further enhanced the degree of wound healing. These iSEPs, isolated by fluorescence reporters, demonstrated even higher clonogenic capacity than primary keratinocytes and were ascertained to be nonmalignant. Further functional analysis of the iSEPs gave rise to epithelial cysts (similar to primary keratinocytes), unlike the behavior of embryonic stem cells and HeLa cells. Whole genomic analysis also shows great similarity between the converted iSEPs and keratinocytes.
This study serves as an initial proof of concept for in vivo regeneration using reprogramming as well as the generation of 3D functional tissue (in mice). This opens the possibilities of exploiting reprogramming science to treat pathologies involving impaired tissue and/or organ repair. (Kurita, M.; et al. Nature
Nanoformulated Curcumin Accelerates Acute Wound Healing through Dkk-1-Mediated Fibroblast Mobilization and MCP-1-Mediated Anti-Inflammation
Severe acute wounds affect more than 300 million people annually, with these numbers expected to increase. Curcumin, the active compound of turmeric, possesses many benefits, including anti-inflammatory, antioxidant, antimicrobial, and anticancer properties. However, its translation into clinical application is impeded due to its hydrophobicity, instability, limited absorption, and quick clearance. Electrospinning is a technique adapted for regenerative medicine and controlled-release systems. To adapt this approach to deliver curcumin for topical wound application, gelatin (rich in peptides and proteins) was spun into nanofibers to deliver curcumin through a biomimetic vehicle for acute wound healing applications.
X-ray diffraction (XRD) spectroscopy showed that curcumin was well dispersed in nanofibrous mats (NMs), with its intense peaks (indicative of purity) reducing intensity. Its endothermic peak from differential scanning calorimetry (DSC) was also lost, suggesting that it was dispersed. Further analysis by Fourier transform infrared spectroscopy (FTIR) confirmed successful incorporation between gelatin and curcumin. The cross-linked structure of the NMs further contributed to sustained curcumin release.
HS-27 fibroblast cultures showed no appreciable difference in lactate dehydrogenase enzyme levels and dead cells (ethidium-homodimer labeling), suggesting biocompatibility. Furthermore, tetrazolium metabolic assays revealed increased activity (reflecting increased cell proliferation) compared with control NMs. Crucially, conditioned media from curcumin-incorporated NMs (Cc/Glt) gave rise to enhanced cell migration by reducing the gap induced by a scratch assay compared with control cultures.
Screening cytokines, chemokines, and growth factors identified six proteins that were significantly downregulated. Further enzyme-linked immunosorbent assays (ELISAs) examined stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) and Wnt signaling pathway activity, through secreted DKK-1 (Dickkopf-related) proteins and SDF-1. Whereas SDF-1 remained unchanged following treatment with CM-Cc/Glt application, Dkk-1 significantly downregulated, as did monocyte chemoattractant protein 1 (MCP-1). Through the addition of blocking antibodies and WAY262611 (a molecular inhibitor of Dkk-1), the enhanced cell migration from CM-Cc/Glt was abrogated. Further studies revealed that the MCP-1 molecule, which regulates inflammatory cell migration, was similarly critical to migration in fibroblasts, monocytes, and macrophages.
In full-thickness wounds generated in rats, CC/Glt NMs resulted in faster wound closure at later time points (days 7, 11, and 15). Histology (hematoxylin and eosin) showed differences in the re-epithelialization and granulation tissue between Cc/Glt NMs and control conditions. In wounds using Glt NM (without curcumin), the wounds were poorly closed, with fibrinous debris observed.
Further observation of collagen in the dermis showed that it was more compact and interwoven, indicating the formation of mature collagen, unlike the loose reticular collagen seen in the control wounds. Moreover, Cc/Glt NM treatment also led to reduced CD68/ED1 macrophage skin infiltration.
As wound dressings, electrospun nanofibrous mats (Glt NMs) can be easily removed from the wound without trauma and isolate microorganisms from the external environment while allowing sufficient gas and fluid exchange. The authors also suggest that Glt NM may help to resolve inflammatory responses related to chronic ulcers (relating to diabetes) and pathological scar formation by, among other things, regulating antioxidants and heme oxygenase activity. Animal models such as streptozotocin-induced diabetic mice or pig wound infection models may allow further optimization of the Cc/Glt NMs for wound healing. Thus, Glt NMs incorporating curcumin serve as mechanically competent carriers to topically deliver drugs to enable wound healing. (Dai, X.; et al. NPG Asia Mater.
Nanomedicine
A Tailored DNA Nanoplatform for Synergistic RNAi/Chemotherapy of Multi-Drug-Resistant Tumors
Multidrug resistance to cancer chemotherapy leads to higher doses of administered drugs because of various physiological cellular mechanisms. P-glycoprotein (Pgp) in resistant cells pumps out drugs, whereas survivin (Sur) inhibits self-regulating apoptotic processes. The authors hypothesize that inhibiting both could circumvent multidrug resistance issues. RNA interference (RNAi), a powerful tool to achieve such knockdown, may be combined with chemotherapy. While siRNA is often used to achieve gene knockdown, small hairpin RNA (shRNA) has comparatively greater stability. Recent studies demonstrated how highly biocompatible DNA nanostructures are suitable for bioimaging, diagnosis, and therapeutics. Addressable DNA origami precisely assemble linear shRNA species. Thus, they have emerged as promising candidates for RNAi and chemo-drug delivery.
The authors employed a facile, universal strategy to load RNAi and chemotherapy drugs into DNA nanostructures. These consisted of triangular DNA origami to carry shRNA against Pgp and Sur. These structures were further augmented to include aptamers targeting mucin 1 (MUC1) and stimuli-responsive cleavable disulfide linkages. This design increased antitumor specificity and synergy against multi-drug-resistant (MDR) tumors.
Using gel electrophoresis, the authors demonstrated efficient assembly of the DNA nanocarrier with cleavable disulfide strands, shPgp, shSur, and targeting aptamers, which exhibited the lowest mobility in 1% agarose gel. This demonstrated successful assembly. This subsequently gave rise to successful loading of doxorubicin (DOX) with efficient release in pH 5.0 or DNase I. Following incubation with MCF-7 DOX-resistant cells, significant DOX accumulation (through its fluorescence signal) was observed compared with free DOX treatment. Further evaluation with fluorescent dye-tagged DNA origami confirmed shRNA release within the cytoplasm. To evaluate successful chemotherapeutic delivery, cell viability decreased to 15% with the DNA nanocarriers while free DOX elicited only 80% viability. This demonstrated DNA nanocarrier efficacy. Further evidence pointed to successful downregulation of Pgp and Sur expression.
A tumor xenograft model was generated in BALB/c nude mice with DOX-resistant MCF-7 cells. DNA nanocarriers containing targeting moieties generated the strongest fluorescence signal with successful tumor targeting. Almost five times more fluorescence signal, indicating greater DOX accumulation, was observed in sectioned tumor tissue. The DNA nanocarrier was further evaluated for its therapeutic efficacy. A dosage of 3 mg/kg DOX and 0.1 mg/kg shPgp and shSur was applied every 6 days for three treatments.
Importantly, the nanocarriers reduced tumor size by silencing Pgp and Sur in vivo, similar to cell culture studies. In addition, DNA nanocarriers also minimized off-target organ toxicity compared with the free drug. In summary, this delivery strategy can be easily adapted for gene-editing systems, antibody drugs, and immunoadjuvants. Such multifunctional systems can open new avenues for cancer therapy development. (Liu, J.; et al. Angew. Chem.
Biomimetic Peptide Display from a Polymeric Nanoparticle Surface for Targeting and Antitumor Activity to Human Triple-Negative Breast Cancer Cells
PEGylated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG NPs) have been used to improve blood circulation and tumor accumulation to enhance chemotherapeutic efficacy. These PLGA-PEG NPs have had relatively little success due to insufficient drug efficacy and accumulation in such off-target tissue as the liver. Improving on passive enhanced permeability and retention (EPR), the cyclic-RGD (cRGD) peptide is utilized to target integrin αvβ3. cRGD ligands increase tumor localization by binding with matrix (extracellular matrix [ECM]) comopnents such as fibronectin and vitronectin. Using a collagen-derived antiangiogenic peptide, AXT050, these are surface functionalized and encapsulated into PLGA-PEG NPs to modulate angiogenesis in an extracellular manner. Surface functionalization and encapsulation of AXT050 increases its loading capacity for NP surface and soluble release to inhibit angiogenesis.
Since the goal of the study was to surface engineer NPs to target tumors and their vasculature, the authors evaluated the binding affinity between recombinant integrin αvβ3 (overexpressed in endothelial cells of the leaky vasculature, tumor, and tumor progenitor cells) and AXT050. The binding affinity between AXT050 and integrin αvβ3 was found to have a kinetic binding curve of 1:1 association and dissociation, which suggests that it is suitable as a targeting moiety.
To evaluate AXT050 tumor tissue localization, near-infrared fluorescence-tagged AXT050, AXT050-IRD800, was intravenously injected into xenografted mice bearing triple-negative breast cancer. AXT050 peptides degraded and cleared from the blood with a half-life of 11 min, although 15% localized to the tumor relative to the other harvested organs (liver, kidneys, spleen, lungs, and heart). A series of PLGA-PEG NPs were formed through nanoprecipitation, allowing AXT050 ligands to coat the particle surface to enable tumor/angiogenic targeting. By varying the mass ratio of conjugated polymer (PLGA-PEG-AXT050) to total polymer (conjugated polymer and methoxy-terminated PLGA-PEG), the NP diameter ranged from 65 to 80 nm, with zeta potential found to be negative.
Fluorescent-labeled NPs also showed a two- to fourfold higher signal in endothelial and human triple-negative breast cancer cells (MDA-MB-231) compared with NPs without targeting peptides. Further analysis with competition assays showed that the interaction between AXT050 peptides and the αvβ3 integrin was critical for NP targeting. Not only did NPs bind with αvβ3 - expressing endothelial cells, but also sustained release of AXT050 peptides further inhibited endothelial and tumor cell proliferation. This was because NPs acted as a reservoir, allowing controlled drug release.
In MDA-MB-231-bearing mice (a model of triple-negative breast cancer), targeted NPs containing different quantities of targeting peptides were observed to have different blood half-lives. The smallest quantity demonstrated the highest half-life, presumably due to greater amounts of PEGylation that decreased clearance. This suggests that optimization is required to strike a balance between delivery quantity and residence time. Among these, 10% NPs (containing 10%/90% of PLGA-PEG-AXT050/PLGA-mPEG polymer) led to the highest accumulation in the breast tumors, extending the half-life to 103 min and accumulating 14% of the peptide dose in an orthotopic mouse model.
This concept to target and disrupt angiogenic supply and tumorigenic cells has promising applications in cancer nanomedicine. (Bressler, E. M.; et al. J. Biomed. Mater. Res. A
Restoration of Tumor-Growth Suppression In Vivo via Systemic Nanoparticle-Mediated Delivery of PTEN mRNA
Occurrence of cancer often involves the increase or mutation of oncogenes alongside the loss of tumor suppression capacity. PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutations are reported in many cancers, including prostate cancer (PCa). PTEN is responsible for generating a dual phosphatase that catalyzes phosphatidylinositol (3,4,5)-trisphosphate dephosphorylation and enhances PI3K-AKT signaling activity. Studies further implicate this PI3K-AKT activity (brought about by PTEN loss) with enhanced tumorigenesis. Thus, suppressing this activity inhibits cancers and sensitizes tumor cells to apoptosis. To date, pharmacological strategies do not fully restore PTEN activity, yet are toxic. Chemically modified mRNA, which does not require nuclear localization and genomic integration, has emerged as a good candidate for gene therapy to restore PTEN function. Yet, its large size, highly negative charge, propensity to degrade, and protein translation capacity have presented challenges in its effective delivery and utilization for therapy.
To overcome this issue, the authors utilized polymer–lipid hybrid nanoparticles (NPs) for systemic delivery of PTEN mRNA to PCa tumors. These hybrid particles were self-assembled using the cationic, lipid-like G0-C14 (complexed with mRNA) and poly(lactic-co-glycolic acid) (PLGA) coated with a lipid-PEG shell. Using a G0-14/mRNA weight ratio of 15 (with little or no mRNA leaching), most mRNA was found to be encapsulated. Spherical NPs were ~120 nm with near-neutral surface charge and NPs stable in serum for 48 h.
PC3 cells (deficient in PTEN) treated with model EGFP-mRNA NPs showed that most of the cells (80%) were still viable at the highest NP concentrations (0.5 µg/mL mRNA) with high viability similar to that observed in DU145 and LNCaP cells. Furthermore, these particles also improved cell transfection efficiency (percentage of GFP+ cells) compared with the commercial transfection agent Lipofectamine 2000. This higher efficiency was similarly observed in other PCa cells—DU145 and LNCaP. Furthermore, NPs protected mRNA species from RNase degradation maintaining consistently high transfection properties (~90%). The NP route of cellular entry and delivery of mRNA cargo was then evaluated. Treatment using macropinocytosis and proton pump inhibitors showed a marked decrease in transfection efficiency, implicating these processes in cellular transport. The NPs were also found to induce a proton-sponge effect to release the cargo in the cytosol. Evidence of cell uptake and endosomal escape underscores the advantages of this NP system.
PTEN mRNA therapeutic candidates were then modified by capping, poly-adenylating the mRNA strands, and tagging them with hemagglutinin (HA) for easy detection and separation. In PC3 cells, the modified PTEN mRNA reduced cell viability and successfully suppressed PI3K-AKT activity. This resulted in PC3 cell viability decrease. The PTEN mRNA NPs were further investigated to ascertain their anticancer efficacy. Treated cells were found to have diminished expression of PI3K-AKT signaling components. These expression levels were even lower than their expression in serum starvation conditions. The number of annexin V-positive cells similarly increased, with four times more cells than control particles (without the payload).
Cell viability in PTEN-deficient cells PCa LNCaP and the LNCaP LN3 subclone was reduced after treatment. In contrast, normal prostate epithelial cells (PreCs) and DU145 cells that were both PTEN competent did not lead to a significant reduction in cell viability. This trend was similarly observed in PTEN-null breast cancer cells, whereas these NPs had no effect on PTEN-competent cells. In vivo pharmacokinetics showed that the NPs could only extend the circulation of naked mRNA to a smaller extent (>5 min), whereas a different formulation extended this to >30 min with ~30% still circulating after 60 min. Importantly, this led to greater NP accumulation on the xenograft tumors and reduced accumulation in the spleen and liver.
To assess their therapeutic efficacy, the reformulated NPs were systemically injected every 3 days with six injections in mice xenografts with PC3 tumors. The PTEN mRNA-bearing NPs gave rise to reduced tumor size without any significant change in weight. Harvested tumors also expressed the highest TUNEL expression (an assay for apoptotic cells) in the PTEN mRNA NPs. To evaluate these in advanced PCa, the PTEN NPs were injected into the mice and evaluated using luciferase-tagged cancer cells. The PTEN NPs resulted in a significant reduction in the number of metastatic lesions (n = 8). To comprehensively evaluate PTEN NPs, they were further tested in a PCa bone model, given that it is the most common site of metastasis. The PC3-luciferase cells were then injected directly into the tibia before the PTEN NPs were injected 5 days after inoculation. Once more, the PTEN NPs reduced the degree of tumor metastasis to a greater extent compared to the control groups. Further examination of the organs and immune status suggests that the effects were mediated by the PTEN NPs instead of any associated immune effects.
This study sheds light on how mRNA can be implemented as a gene therapeutic. While PTEN loss has been recognized for >2 decades as causing PCa progression, the advances in delivery vehicle generation and payload development have enabled this proof of concept for gene “gain of function” in PCa therapy. These findings may lead to the development of gene therapy involving other tumor suppressors, such as p53, that have proven difficult to translate. (Islam, M. A.; et al. Nat. Biomed. Eng.
Neutrophil Membrane-Coated Nanoparticles Inhibit Synovial Inflammation and Alleviate Joint Damage in Inflammatory Arthritis
Rheumatoid arthritis is a deadly autoimmune disease with systemic inflammation symptoms that result in joint problems. Although biologics have shown promise, they carry numerous limitations. Since it involves a large number of molecular players, single-molecule approaches may not be able to impede disease progression. Current treatments achieve sustained clinical remission and 30% of the population is poorly controlled.
Alternative anti-inflammatory approaches have become highly desirable due to these inadequacies. A recent promising approach has involved fusing natural cell membranes onto synthetic cores. The cell-based covering (containing the antigenic profile of the source cells) can absorb and neutralize pathological molecules. This approach has been previously explored by this group using red blood cell (RBC) membrane nanoparticles (NPs) to bind toxins.
This approach was then adapted to create neutrophil-like NPs as anti-inflammatory agents to manage rheumatoid arthritis. These function by binding with immune-regulatory molecules that would have otherwise targeted endogenous neutrophils.
Activated plasma membrane obtained from human peripheral blood neutrophils were coated on poly(lactic-co-glycolic acid) (PLGA) polymeric core particles. The coating increased the particle by ~18 nm, enriched with key surface antigens such as TNF-α receptor (TNF-αR), IL-1 receptor (IL-1R), and lymphocyte function-associated antigen (LFA-1).
In chondrocytes activated with TNF-α and human umbilical vein endothelial cells (HUVECs), significantly higher quantities of neutrophil-NPs were observed to attach compared with RBC-NPs. This demonstrates the greater affinity of neutrophil-NPs than RBC-NPs with activated chondrocytes. Incubation with 10 ng/mL IL-1B or 100 ng/ mL TNF-α led to chondrocyte activation (indicated by increased ICAM-1 expression). IL-1β or TNF-α incubation caused 80% of cells to undergo apoptosis, but neutrophil-NPs reduced this fraction, leading to increases in IC50 values for IL-1β and TNF-α.
These were similarly repeated in human synovial fluid (hSF) from patients suffering from rheumatoid arthritis. Once more, neutrophil-NPs significantly reduced the rate of chondrocyte apoptosis and HUVEC activation by hSF. The neutrophil-NPs also prevented aggrecan degradation of hSF-activated chondrocytes. Using a mouse model to evaluate neutrophil-NP penetration of cartilage, femoral heads were cultured in medium supplemented with IL-1β to mimic inflammatory arthritis. Neutrophil-NPs were found to penetrate the surface cartilage matrix with decaying signal upon increasing penetration depth. This dropped to 1% at 140 μm depth, implying intracellular uptake by stimulated chondrocytes.
When these femoral heads were exposed to IL-1β in culture medium, matrix fibrillation (cartilage loss) was observed to have extended down into the midzone. Whereas RBC-NPs had a similar morphology to IL-1β treatment, neutrophil-NPs halted the damage induced by IL-1β. Tissues treated with IL-1β alone or supplemented with RBC-NPs only had 40% of cartilage remaining (safranin-O staining), while IL-1β and neutrophil-NPs maintained more than 80% of the original cartilage. This chondroprotective result was mediated by the neutralization of the arthritic factors.
Neutrophil-NPs were then evaluated on a murine model of collagen-induced arthritis (CIA). The neutrophil-NP application led to smaller knee diameters compared with the phosphate-buffered saline (PBS) group (reduced cellular influx and edema formation) with equivalent knee sizes treated with neutralizing antibodies (IL-1β and TNF-α). Whereas the neutrophil-NP group had an even distribution of chondrocytes in the articular cartilage (no obvious degeneration), the PBS-treated group displayed intense neutrophil infiltration in the joints and synovium.
Fibroblast-like synoviocytes (FLs), an invasive cell type (commonly occurring in rheumatoid arthritis) that stains for CD248 and fibronectin in the synovial intimal lining, were also examined. The neutrophil-NPs largely suppressed this phenotype, whereas they were strongly observed in the PBS-treated group.
This was further transferred to a transgenic mouse model expressing human TNF-α transgenes that spontaneously develop arthritis. Once more, the neutrophil-NPs preserved the chondrocytes at a similar level to the group treated with blocking TNF-α antibodies and similar levels of FLs.
In this CIA model, a prophylactic regimen was implemented, with neutrophil-NP treatment to prevent arthritic induction. Once more, the neutrophil-NPs proved effective to reduce knee diameter, coinciding with reduced immune infiltration and preservation of cartilage. A fascinating observation from this study was the distant neutralizing effects afforded by the neutrophil-NPs. They appeared to neutralize arthritogenic factors and elicit a systemic therapeutic response. The serum levels of TNF-α and IL-1β (that correlate with rheumatoid arthritis severity) strongly increased. Furthermore, the systemic effects of neutrophil-NPs were seen to ameliorate the symptoms observed in the mouse ankle and paw (volume, swelling, and redness), resulting in a lower arthritis score than PBS treatment.
This study demonstrates the potential of using cell membrane-coated NPs to treat diseases, particularly rheumatoid arthritis, that involve numerous cell types that interplay with diffusible factors to give rise to systemic responses.
While this technology was promising during proof of concept, clinical translation of neutrophil-NPs is not straightforward, as natural cell membranes are required for technology development. Thus, ex vivo production of neutrophils with universal immunity at a clinical scale will be required for clinical studies. (Zheng, Q.; et al. Nat. Nanotechnol.
Nanotechnology
Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA In Vivo Induces Homology-Directed DNA Repair
CRISPR/Cas9 gene-editing systems often exploit homology-directed repair (HDR) to correct mutated genes back to their wild-type sequence. For HDR therapy, in vivo editing is challenging because the components of Cas9, gRNA, and donor DNA need to be effectively delivered. Adeno-associated viruses (AAVs) are the most advanced method for Cas9 delivery in vivo, although a large fraction of humans have preexisting immunity that makes them ineligible candidates. They also cause off-target genomic damage and have a small packing size (multiple viruses needed to deliver the components), which reduces HDR efficiency. Moreover, the viral titers required for editing are typically orders of magnitude higher than clinically acceptable levels.
An alternative is to use nonviral vehicles for direct delivery: lipofectamine (lipid molecules) and polyethylenimine (PEI; cationic reagents). These are potentially problematic due to the delivery of multiple macromolecules in vivo. In this study, a delivery platform, termed CRISPR-Gold, was utilized for the direct delivery of Cas9 and donor DNA. It comprises gold nanoparticles conjugated with DNA, complexed with donor DNA, Cas9, and PAsp(DET)—an endosomal disruptive polymer. PAsp(DET), the cationic component, triggers endosomal disruption after cell endocytosis before releasing CRISPR-Gold into the cytoplasm before glutathione releases the DNA payload.
CRISPR-Gold circumvents the key challenge of delivering proteins and nucleic acids by simultaneously complexing both. The gold nanoparticle core allows DNA to be densely packed and delivered into multiple cell types.
It displayed a 61.5% encapsulation efficiency, while still maintaining its enzymatic activity. Using HEK293 cells, experiments were performed to express blue fluorescence to verify HDR induction in cells.
CRISPR-Gold was utilized containing a single-stranded donor oligonucleotide (ssODN) that converted blue fluorescent protein (BFP) into the green fluorescent protein (GFP) gene and a gRNA that cuts the BFP gene. This induced 11.3% of the BFP-HEK cells to express GFP via HDR, which was further confirmed using sequencing. Investigating dose response, CRISPR-Gold exhibited a maximum HDR efficiency at 8 µg/mL of Cas9. At higher concentrations, HDR efficiency was reduced due to increased cell toxicity. Further investigations suggest that caveolae/raft-dependent endocytosis was responsible for the uptake of CRISPR-Gold.
Its uptake was then evaluated in a panel of therapeutically relevant cells, including human embryonic stem (hES) cells, human induced pluripotent stem (hiPS) cells, primary bone marrow-derived dendritic cells (BMDCs), and primary myoblasts.
CRISPR-Gold was then synthesized to edit the dystrophin or CXCR4 gene at an HDR efficiency of 3%–4%. These were considerably less toxic than lipofectamine or nucleofection methods. Mouse experiments were then performed to evaluate if Cas9 could be delivered in vivo. These generated fluorescent proteins to indicate gene deletions following intramuscular injections of CRISPR-Gold. After 2 weeks, fluorescence expression was seen in the muscle sections demonstrating the in vivo delivery of Cas9 for gene editing. Duchenne muscular dystrophy (DMD) was then chosen as the initial application using mdx mice, which do not express dystrophin. CRISPR-Gold corrected the mutated dystrophin gene after a single injection, restoring dystrophin protein expression in the muscle tissue. A total of 5.4% of the dystrophin gene was corrected—~18-fold higher than without using the delivery vehicle (Cas9 RNP and donor DNA). CRISPR-Gold led to reduced muscle fibrosis, indicative of better tissue health.
The treated mice were subjected to functional tests, namely, the four-limb hanging test. CRISPR-Gold treatment increased hanging time by twofold compared with mice given a noncoding sequence. Its off-target DNA damage was also found to be expressed at background levels through genome sequencing. Immunogenic response was also evaluated by injecting CRISPR-Gold (6 mg/kg Cas9 protein) and serial injections. Although a greater presence of CD45+ and CD11b cells was observed (macrophages), the systemic/plasma cytokine profile and subject weight suggested minimal immunogenicity, that it could be safely used.
In summary, the authors demonstrated that CRISPR-Gold is a suitable nonviral delivery platform that enables in vitro and in vivo gene editing via HDR. It has the potential to restore the mutation back to its wild-type status, fully regenerating dystrophin to treat DMD in animal models. This is a proof of concept for treating genetic diseases. (Lee, K.; et al. Nat. Biomed. Eng.
CRISPR/Cas9 Genome Editing Using Gold Nanoparticle-Mediated Laserporation
CRISPR/Cas9 specificity is due to CRISPR RNA (crRNA) bound to the enzyme by trans-activating crRNA (tracrRNA). Currently there is a lack of efficient, transient carriers to deliver CRISPR/Cas9 enzymes. To circumvent such limitations, the authors utilized the gold nanoparticle-mediated (GNOME) laserporation technique that combines nanotechnology and laser physics. Following short-pulsed laser irradiation (of gold nanoparticles), nanobubbles are formed by heating in nanoseconds, that permeabilize the cells for small molecules to diffuse in. Using previously established parameters, GNOME laserporation successfully delivers mCCR7 gRNA into Cas9 and mCCR7-expressing cells that possess gene-editing capability.
Seventy-kilodalton fluorescein isothiocyanate (FITC) dextrans were incubated with 200 nm gold nanoparticles and assessed within 1 h after GNOME laserporation. This was found to increase the FITC+ cells by 13%–38%. GNOME laserporation also had negligible impact on cell viability. GNOME laserporation was then applied to primary, hard-to-transfect cells—in vitro-activated mouse CD8+ T cells. The mouse chemokine receptor CXCR3 (mCXCR3) was used as a model gene for editing. The technique generated a knockout efficacy reaching 19.1% of all cells 3 days after laserporation. Using cultured lymph node stroma cells, the authors further knocked out enhanced green fluorescent protein (EGFP), knocking it out in up to 4.7% cells. This demonstrates the suitability of GNOME laserporation for editing a variety of different cells in vitro.
In summary, this approach can avoid cloning and packaging Cas9 and/or gRNA into viral particles. Moreover, it reached a throughput of 1 well of a 96-well plate in ~10 s, even allowing the delivery of commercially available gRNAs. Overall, it was also found to be easier to use compared with electroporation and lipofection methods. (Bošnjak, B.; et al. Adv Biosyst.
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.