Physiologically based modeling of LNP-mediated delivery of mRNA in the vascular system.

RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic (PK)-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically based PK/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles (LNPs) in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue PKs of LNPs, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within a 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing LNPs developed for an array of conditions and across species.

In vivo hematopoietic stem cell modification by mRNA delivery.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

3D-printed bi-layered polymer/hydrogel construct for interfacial tissue regeneration in a canine model.

OBJECTIVES: There are complications in applying regenerative strategies at the interface of hard and soft tissues due to the limited designs of constructs that can accommodate different cell types in different sites. The problem originates from the challenges in the adhesion of dissimilar materials, such as polymers and hydrogels, that can be suitable for regenerating different tissues such as bone and soft tissues. This paper presents a design of a new hybrid construct in which a polymer (polycaprolactone (PCL)) membrane firmly adheres to a layer of hydrogen (gelatin). METHODS: PCL membranes with defined size and porosity were fabricated using 3D printing. The gelatin layer was attached to the PCL membranes using the aminolysis procedure. We have examined this construct for the application of Guided Bone Regeneration (GBR) as a typical surgical regenerative procedure of the oral cavity at the interface of bone and soft tissue. Complete in vitro and in vivo investigations on canine tibia bone defects have been performed. Histological analyses for fibrosis morphometric and bone morphometric evaluation, as well as bone-fibrosis histological grading and CBCT imaging, were conducted. RESULTS: Chemical and morphological studies of the membrane proved that gelatin was uniformly attached to the aminolyzed PCL membranes. The in vitro and in vivo studies indicated the membrane's biocompatibility, mechanical stability, and barrier function for the GBR application. Furthermore, in vitro study showed that the membranes could improve osteogenesis and the regeneration of bone defects. The results illustrated that the mean bone density in the membrane groups was about three times more than that of the control group. SIGNIFICANCE: The fabricated 3D-printed hybrid Gelatin/PCL bi-layered membrane can be a good candidate for interfacial tissue engineering and a promising membrane for GBR procedure.

Development of a Modular Reinforced Bone Tissue Engineering Scaffold with Enhanced Mechanical Properties.

A modular design composed of 3D-printed polycaprolactone (PCL) as the load-bearing module, and dual porosity gelatin foam as the bio-reactive module, was developed and characterized in this study. Surface treatment of the PCL module through aminolysis-aldehyde process was found to yield a stronger interface bonding compared to NaOH hydrolysis, and therefore was used in the fabrication procedure. The modular scaffold was shown to significantly improve the mechanical properties of the gelatin foam. Both compressive modulus and ultimate strength was found to increase over 10 times when the modular design was employed. The bio-reactive module i.e., gelatin foam, presented a dual porosity network of 100-300 µm primary and <10 µm secondary pores. SEM images revealed excellent attachment of DPSCs to the bio-reactive module.

CAR T cells produced in vivo to treat cardiac injury.

Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified messenger RNA (mRNA) in T cell­targeted lipid nanoparticles (LNPs). The efficacy of these in vivo­reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.

In vivo efficacy of 3D-printed elastin-gelatin-hyaluronic acid scaffolds for regeneration of nasal septal cartilage defects.

Nasal septal cartilage perforations occur due to the different pathologies. Limited healing ability of cartilage results in remaining defects and further complications. This study sought to assess the efficacy of elastin-gelatin-hyaluronic acid (EGH) scaffolds for regeneration of nasal septal cartilage defects in rabbits. Defects (4 × 7 mm) were created in the nasal septal cartilage of 24 New Zealand rabbits. They were randomly divided into four groups: Group 1 was the control group with no further intervention, Group 2 received EGH scaffolds implanted in the defects, Group 3 received EGH scaffolds seeded with autologous auricular chondrocytes implanted in the defects, and Group 4 received EGH scaffolds seeded with homologous auricular chondrocytes implanted in the defects. After a 4-month healing period, computed tomography (CT) and magnetic resonance imaging (MRI) scans were obtained from the nasal septal cartilage, followed by histological evaluations of new tissue formation. Maximum regeneration occurred in Group 2, according to CT, and Group 3, according to both T1 and T2 images with 7.68 ± 1.36, 5.44 ± 2.41, and 8.72 ± 3.02 mm2 defect area respectively after healing. The difference in the defect size was statistically significant after healing between the experimental groups. Group 3 showed significantly greater regeneration according to CT scans and T1 and T2 images. The neocartilage formed over the underlying old cartilage with no distinct margin in histological evaluation. The EGH scaffolds have the capability of regeneration of nasal cartilage defects and are able to integrate with the existing cartilage; yet, they present the best results when pre-seeded with autologous chondrocytes.

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg-1, or intravenously (IV), 2 mg kg-1, and then IV administer modmRNA-LNP, 0.32 mg kg-1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.

Disseminated Intravascular Coagulation Associated with Large Deletion of Immunoglobulin Heavy Chain.

Although the majority of monogenic defects underlying primary immunodeficiency are microlesions, large lesions like large deletions are rare and constitute less than 10% of these patients. The immunoglobulin heavy chain (IGH) locus is one of the common regions for such genetic alterations. This study describes a rare case of autosomal recessive agammaglobulinemia with a homozygous large deletion in chromosome 14q32.33 (106067756-106237742) immunoglobulin heavy chain clusters with an unusual and severe skin infection and disseminated intravascular coagulopathy.

Osteo-mucosal engineered construct: In situ adhesion of hard-soft tissues.

OBJECTIVES: The aim of this work was to combine engineered hard and soft tissue, adopting a new method for interfacial adhesion of osteo-mucosal construct. We hypothesized that the chemical procedure involved in this method not only adheres the components, but also improves the cell growth inside them. METHODS: 3D-printed functionally-graded porous hard-tissue scaffolds were characterized, functionalized by aminolysis and tyrosinase, and accommodated by human osteoblast cells. Introducing amino groups through aminolysis and inducing dopaquinones by tyrosinase can take part in the Michael additions to cause the adhesion. Subsequently, fully-differentiated engineered oral mucosa was formed directly on the surface of hard tissue. Constructs were assessed in term of morphology, structure, chemical composition, histology, and cytocompatibility. Interfacial adhesion was compared to a control group prepared by using a biological glue for the attachment of the soft and hard tissues. RESULTS: The data confirmed higher proliferation of osteoblast cells via aminolysis and improved osteoblast cells distribution and differentiation by incorporation of tyrosinase in collagen. There was evidence of multilayered, stratified epithelium on the osteo-mucosal model with viable fibroblasts and osteoblasts within the lamina propria and bone tissue layers. Our method of adhesion resulted in cohesive debonding within the engineered soft tissue; while in the control group, adhesive debonding and complete separation of the oral mucosa from the hard tissue was observed. Although the shear strength of the osteo-mucosal model (157.6 kDa ± 25.1) was slightly higher than that of the control group (149.4 kDa ± 23.1), there was no statistically significant difference between them (p > 0.05). However, the advantage of our in situ adhesion approach is the absence of a barrier like glue which can disrupt direct cellular communications between tissues. SIGNIFICANCE: This study provides a novel method of directly combining tissue-engineered human bone with oral mucosa, which has the potential to improve cell-ingrowth and tissue integration. This engineered tissue construct, after further optimization, can be used clinically as a graft material in various oral surgeries and can also be employed as an in vitro model to investigate many aspects of oral diseases and examine dental materials and oral health care products as a replacement of in vivo models.

Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs.

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Theranostic applications of stimulus-responsive systems based on carbon dots.

Over recent years, many different nanoparticle-based drug delivery systems (NDDSs) have been developed. Recently the development of stimulus-responsive NDDSs has come into sharper focus. Carbon dots (CDs) possess outstanding features such as useful optical properties, good biocompatibility, and the ability for easy surface modification. Appropriate surface modification can allow these NDDSs to respond to various chemical or physical stimuli that are characteristic of their target cells or tissue (frequently malignant cells or tumors). The present review covers recent developments of CDs in NDDSs with a particular focus on internal stimulus response capability that allows simultaneous imaging and therapeutic delivery (theranostics). Relevant stimuli associated with tumor cells and tumors include pH levels, redox potential, and different enzymatic activities can be used to activate the CDs at the desired sites.

Pressure-Assisted Coating of Ceramics on 3D-Printed Polymeric Scaffolds.

Pressure-assisted coating (PAC) is introduced to coat 3D-printed polymeric scaffolds with ß-tricalcium phosphate (ß-TCP) for tissue engineering applications. The method consists of four steps: infiltration of ceramic particles into the porous structure of the polymeric scaffold, dehydration of the slurry, compaction of ceramic particles around the scaffold, and heat treatment. The optimal coating is obtained at an infiltration speed of 400 mm/min followed by complete dehydration, compaction under ca. 8 MPa pressure, and subsequent heat treatment at 65 °C. The outcome is a uniformly coated scaffold with no deformation or structural defects, as confirmed by micro-CT analysis and laser and scanning electron microscopy. Scaffolds coated using the PAC method present superior interface bonding strength compared to those coated with a biomimetic approach. The contact angle decreased from 75.2 ± 1.4° for the uncoated scaffold to 39.6 ± 9.6° for the PAC specimen. PAC also increased the surface roughness from 0.66 ± 0.08 to 6.89 ± 0.26 µm and doubled the number of attached cells on the 3rd day of culture. The described method is applicable to different structures, object sizes, pore sizes, and shapes. For instance, in-depth coating of a 10 mm × 10 mm (D × H) cone with a 58 ± 4 µm-thick layer of ß-TCP can be achieved using PAC. The method can be used to coat other polymers, such as poly(lactic-co-glycolic acid) (PLGA). Successful coating of ß-TCP on 3D-printed PLGA scaffolds is also presented as a proof of concept.

Development of an anti-CD45RA-quantum dots conjugated scFv to detect leukemic cancer stem cells.

Leukemic cancer stem cells (LSCs), aberrantly overexpressing CD45RA are among the major causes of relapse following chemotherapy in patients with acute myeloid leukemia and serve as a highly sensitive marker for predicting relapse occurrence following chemotherapy. The main purpose of current study was to develop a sensitive approach for detecting LSCs based on a conjugate of an anti-CD45 scFv and quantum dot. The variable light and heavy chain sequences of a recently developed anti-CD45RA monoclonal antibody were derived from hybridoma cells and PCR amplified to construct scFv. Following insertion of scFv gene into a pET32a-lic vector and expression in Escherichia coli and purification, the purified scFv, was conjugated with carbon dots (C dots) and used for the detection of CD45RA +cells while CD45RA-cells served as negative control. Subsequently, Functional activity of the conjugate was analyzed by flow cytometry and ICC to detect the cell surface antigen binding and detection ability. Based on results, purified CD45RA scFv conjugated C dots could specifically recognize CD45RA positive cells, but not any CD45RA negative ones. In conclusion, here we developed a low-cost but very efficient approach for detection of CD45RA positive cells including LSCs.

Bottom-up synthesis of nitrogen and oxygen co-decorated carbon quantum dots with enhanced DNA plasmid expression.

In this paper, a bottom-up hydrothermal route is reported for the synthesis of oxygen and nitrogen co-decorated carbon quantum dots (CQDs) using ammonium hydrogen citrate (AHC) as a single precursor. DLS data approved the formation of 4.0 nm (average size) CQDs. XRD pattern shows the interlayer spacing (002) of 3.5 Šfor CQDs, which is exactly the same as that of crystalline graphite. XPS and FTIR spectra verified the formation of oxygen and nitrogen functional groups on the CQDs surface. Co-decorated carboxyl, hydroxyl and amine groups on the CQDs surfaces make them as promising polyelectrolyte for gene delivery. Toxicity assay showed a survival rate of 70% under different incubation times and up to 500 µg/mL. The highly water-soluble, stable fluorescence and low toxic CQDs increased the gene expression of DNA plasmid in E. coli bacteria 4-fold more than the control group.

Self-assembling of graphene oxide on carbon quantum dot loaded liposomes.

This paper describes the design of stimuli-sensitive theranostic nanoparticles, composed of reduced graphene oxide (rGO) self-assembled on thermosensitive liposomes encapsulated doxorubicin (DOX) and carbon quantum dot (CQD) (CQD-DOX-rGO-Tlip). The rGO-Tlip particles have been observed to be flower-shaped objects. The thermoresponsive and theranostic potential of CQD-DOX-rGO-Tlips have been studied using differential scanning calorimetry (DSC), ultraviolet visible spectroscopy (UV-Vis), Raman spectroscopy and photoluminescent assays. The chemo-photothermal potential of rGO-Tlip on MD-MB-231 cells during NIR laser irradiation has been examined using MTT assay. Also, the ability of rGO-Tlip to be taken up by MD-MB-231 cells has been studied using confocal microscopy and flowcytometry. The results indicate that CQD-DOX-rGO-Tlips achieve a synergistic effect between photothermal therapy and chemotherapy for cancer treatment. Furthermore, online monitoring drug release is accomplished by studying the emission intensity of CQD while DOX released.

An electrochemical biosensor for prostate cancer biomarker detection using graphene oxide-gold nanostructures.

In this paper, a most sensitive electrochemical biosensor for detection of prostate-specific antigen (PSA) was designed. To reach the goal, a sandwich type electrode composed of reduced graphene oxide/ gold nanoparticles (GO/AuNPs), Anti-Total PSA monoclonal antibody, and anti-Free PSA antibody was assembled. The functionalized materials were thoroughly characterized by atomic force microscope spectroscopy, transmission electron microscopy, and X-ray diffraction techniques. The electrochemical properties of each of the modification step were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. The results presented that the proposed biosensor possesses high sensitivity toward total and free PSA. Furthermore, the fabricated biosensor revealed an excellent selectivity for PSA in comparison to the other tumor markers such as BHCG, Alb, CEA, CA125, and CA19-9. The limit of detection for the proposed electrochemical biosensor was estimated to be around 0.2 and 0.07 ng/mL for total and free PSA antigen, respectively.

Dual Porosity Protein-based Scaffolds with Enhanced Cell Infiltration and Proliferation.

3D dual porosity protein-based scaffolds have been developed using the combination of foaming and freeze-drying. The suggested approach leads to the production of large, highly porous scaffolds with negligible shrinkage and deformation compared to the conventional freeze-drying method. Scanning electron microscopy, standard histological processing and mercury intrusion porosimetry confirmed the formation of a dual network in the form of big primary pores (243 ± 14 µm) embracing smaller secondary pores (42 ± 3 µm) opened onto their surface, resembling a vascular network. High interconnectivity of the pores, confirmed by micro-CT, is shown to improve diffusion kinetics and support a relatively uniform distribution of isolated human dental pulp stem cells within the scaffold compared to conventional scaffolds. Dual network scaffolds indicate more than three times as high cell proliferation capability as conventional scaffolds in 14 days.

A genosensor for detection of HTLV-I based on photoluminescence quenching of fluorescent carbon dots in presence of iron magnetic nanoparticle-capped Au.

Carbon dots and Fe3O4@Au were synthesized to develop a new biosensor to detect DNA target. We investigated the photoluminescence property of carbon dots (CDs) in the presence of Fe3O4-capped Au (Fe3O4@Au). Firstly, we designed two dedicated probes for unique long sequence region of human T-lymphotropic virus type 1 genome. One of the probes was covalently bound to the CDs. In the absence of target, CDs-probe was adsorbed on the surface of Fe3O4@Au through two possible mechanisms, leading to quenching the fluorescence emission of CDs. The fluorescence emission of CDs was recovered in the presence of target since double-stranded DNA cannot adsorb on the Fe3O4@Au. Also, Fe3O4@Au can adsorb the unhybridized oligonucleotides and improves the accuracy of detection. The specificity of the proposed biosensor was confirmed by BLAST search and assessed by exposing the biosensor to other virus targets. The experimental detection limit of the biosensor was below 10 nM with linear range from 10 to 320 nM.

Optimizing the nanostructure of graphene oxide/silver/arginine for effective wound healing.

In this study, we introduce a novel graphene oxide/silver/arginine (GO/Ag/Arg) nanohybrid structure, which can act as an angiogenesis promoter and provide antibacterial nanostructure for improving the wound healing process. GO/Ag nanostructure has been optimized in terms of the GO/Ag mass ratio and pH values using central composite design and the response surface method to increase the Ag loading efficiency. Then, Arg was chemically introduced to the surface of GO/Ag nanostructure. Electrospun polycaprolactone (PCL)-GO/Ag/Arg nanocomposite was successfully fabricated and characterized. The synthesized nanocomposite demonstrated not only a great antibacterial effect on both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacterial species, but appropriate biocompatibility against L929 fibroblastic cell lines. The results demonstrated that the preparation of the PCL-GO/Ag/Arg nanocomposite at a concentration of 1.0 wt% GO/Ag/Arg possessed the best biological and mechanical features. In vivo experiments also revealed that the use of optimized PCL-GO/Ag/Arg nanocomposite, after 12 d of treatment, led to significant increase in the healing process and also regeneration of the wound via reconstruction of a thickened epidermis layer on the wound surface, which was confirmed by histological analysis. In conclusion, the proposed approach can introduce a novel notion for preparing antibacterial material that significantly promotes angiogenesis.

Early diagnosis of disease using microbead array technology: A review.

Early diagnosis of diseases (before they become advanced and incurable) is essential to reduce morbidity and mortality rates. With the advent of novel technologies in clinical laboratory diagnosis, microbead-based arrays have come to be recognized as an efficient approach, that demonstrates useful advantages over traditional assay methods for multiple disease-related biomarkers. Multiplexed microbead assays provide a robust, rapid, specific, and cost-effective approach for high-throughput and simultaneous screening of many different targets. Biomolecular binding interactions occur after applying a biological sample (such as blood plasma, saliva, cerebrospinal fluid etc.) containing the target analyte(s) to a set of microbeads with different ligand-specificities that have been coded in planar or suspension arrays. The ligand-receptor binding activity is tracked by optical signals generated by means of flow cytometry analysis in the case of suspension arrays, or by image processing devices in the case of planar arrays. In this review paper, we discuss diagnosis of cancer, neurological and infectious diseases by using optically-encoded microbead-based arrays (both multiplexed and single-analyte assays) as a reliable tool for detection and quantification of various analytes.