Optimized rod length of polyplex micelles for maximizing transfection efficiency and their performance in systemic gene therapy against stroma-rich pancreatic tumors

Poly(ethylene glycol) (PEG) modification onto a gene delivery carrier for systemic application results in a trade-off between prolonged blood circulation and promoted transfection because high PEG shielding is advantageous in prolonging blood retention, while it is disadvantageous with regard to obtaining efficient transfection owing to hampered cellular uptake. To tackle this challenging issue, the present investigation focused on the structure of polyplex micelles (PMs) obtained from PEG–poly(l-lysine) (PEG–PLys) block copolymers characterized as rod-shaped structures to seek the most appreciable formulation. Comprehensive investigations conducted with particular focus on stability, PEG crowdedness, and rod length, controlled by varying PLys segment length, clarified the effect of these structural features, with particular emphasis on rod length as a critical parameter in promoting cellular uptake. PMs with rod length regulated below the critical threshold length of 200 nm fully exploited the benefits of cross-linking and the cyclic RGD ligand, consequently, exhibiting remarkable transfection efficiency comparable with that of ExGen 500 and Lipofectamine^® LTX with PLUS™ even though PMs were PEG shielded. The identified PMs exhibited significant antitumor efficacy in systemic treatment of pancreatic adenocarcinoma, whereas PMs with rod length above 200 nm exhibited negligible antitumor efficacy despite a superior blood circulation property, thereby highlighting the significance of controlling the rod length of PMs to promote gene transduction.     

FRET-enabled biological characterization of polymeric micelles

Translation of micelles from the laboratory to the clinic is limited by a poor understanding of their in vivo fate following administration. In this paper, we establish a robust approach to real-time monitoring of the in vivo stability of micelles using Förster Resonance Energy Transfer (FRET). This characterization method allows for exquisite insight into the fate of micellar constituents, affording the capabilities to rapidly and efficiently evaluate a library of synthetically derived micellar systems as new therapeutic platforms in vivo. FRET-enabled biological characterization further holds potential to tailor material systems being uniquely investigated across the delivery community towards the next generation of stable therapeutics for disease management.     

Water-dispersible magnetic carbon nanotubes as T2-weighted MRI contrast agents

An efficient MRI T₂-weighted contrast agent incorporating a potential liver targeting functionality was synthesized via the combination of superparamagnetic iron oxide (SPIO) nanoparticles with multiwalled carbon nanotubes (MWCNTs). Poly(diallyldimethylammonium chloride) (PDDA) was coated on the surface of acid treated MWCNTs via electrostatic interactions and SPIO nanoparticles modified with a potential targeting agent, lactose–glycine adduct (Lac–Gly), were subsequently immobilized on the surface of the PDDA–MWCNTs. A narrow magnetic hysteresis loop indicated that the product displayed superparamagnetism at room temperature which was further confirmed by ZFC (zero field cooling)/FC (field cooling) curves measured by SQUID. The multifunctional MWCNT-based magnetic nanocomposites showed low cytotoxicity in vitro to HEK293 and Huh7 cell lines. Enhanced T₂ relaxivities were observed for the hybrid material (186 mm⁻¹ s⁻¹) in comparison with the pure magnetic nanoparticles (92 mm⁻¹ s⁻¹) due to the capacity of the MWCNTs to “carry” more nanoparticles as clusters. More importantly, after administration of the composite material to an in vivo liver cancer model in mice, a significant increase in tumor to liver contrast ratio (277%) was observed in T₂ weighted magnetic resonance images.     

The in vitro and in vivo toxicity of graphene quantum dots

Graphene quantum dots (GQD) generate intrinsic fluorescence, and improves aqueous stability of graphene oxide (GO) while maintaining wide chemical adaptability and high adsorption capacity. Despite GO's remarkable advantages in bio-imaging, bio-sensing and other biomedical applications, its biosafety issues are still unclear. Here we report a detailed and systematic study on the in vitro and in vivo toxicity of GQD. The GQD sample was prepared through a facile oxidation approach and fully characterized by means of AFM, TEM, FTIR, XPS and elemental analysis. In vitro experiments showed that GQD exhibits very low cytotoxicity owing to its ultra-small size and high oxygen content. Then, the in vivo biodistribution experiment of GQD revealed no material accumulation in main organs of mice and fast clearance of GQD through kidney. In order to mimic clinic drug administration, mice were injected with GQD and GO (as comparison) multiple times for in vivo toxicity tests. We found that GQD showed no obvious influence on mice owing to its small size, while GO appeared toxic, even caused death to mice due to GO aggregation inside mice. In brief, GQD possesses no obvious in vitro and in vivo toxicity, even under multi-dosing situation.     

Near-infrared fluorescence imaging using organic dye nanoparticles

Near-infrared (NIR) fluorescence imaging in the 700–1000 nm wavelength range has been very attractive for early detection of cancers. Conventional NIR dyes often suffer from limitation of low brightness due to self-quenching, insufficient photo- and bioenvironmental stability, and small Stokes shift. Herein, we present a strategy of using small-molecule organic dye nanoparticles (ONPs) to encapsulate NIR dyes to enable efficient fluorescence resonance energy transfer to obtain NIR probes with remarkably enhanced performance for in vitro and in vivo imaging. In our design, host ONPs are used as not only carriers to trap and stabilize NIR dyes, but also light-harvesting agent to transfer energy to NIR dyes to enhance their brightness. In comparison with pure NIR dyes, our organic dye nanoparticles possess almost 50-fold increased brightness, large Stokes shifts (∼250 nm) and dramatically enhanced photostability. With surface modification, these NIR-emissive organic nanoparticles have water-dispersity and size- and fluorescence- stability over pH values from 2 to 10 for almost 60 days. With these superior advantages, these NIR-emissive organic nanoparticles can be used for highly efficient folic-acid aided specific targeting in vivo and ex vivo cellular imaging. Finally, during in vivo imaging, the nanoparticles show negligible toxicity. Overall, the results clearly display a potential application of using the NIR-emissive organic nanoparticles for in vitro and in vivo imaging.     

Reduction of ectopic bone growth in critically-sized rat mandible defects by delivery of rhBMP-2 from kerateine biomaterials

Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.     

A 5-fluorouracil-loaded polydioxanone weft-knitted stent for the treatment of colorectal cancer

In-stents restenosis caused by tumour ingrowth is a major problem for patients undergoing stent displacement because the conventional stents often lack a sustained anti-tumour capability. The aim of this paper was to develop a weft-knitted polydioxanone stent which can slow release 5-fluorouracil (5-FU). In order to determine the most suitable drug concentration, the 5-FU safe concentration in vivo and appropriate loading percentage in the membranes were investigated, and then 5-FU-loaded poly-l-lactide membranes at concentration of 3.2%, 6.4% and 12.8% were coated onto the stent using electro-spinning method, respectively. The morphology, chemical structure and in vitro drug release property of the coating membranes were subsequently examined. Their anti-tumour activity and mechanism were assessed in vitro and in vivo using a human colorectal cancer cell line HCT-116 and tumour-bearing BALB/c nude mice. The half maximal inhibitory concentration (IC₅₀) and the median lethal dose (LD₅₀) demonstrated that the 6.4% and 12.8% membranes had better anti-tumour effects than pure 5-FU due to the sustainable drug releasing property of the coated membranes on the stent. The membranes possessing appropriate drug loading doses, such as 6.4% or 12.8% also provided better anti-in-stents restenosis effects than other groups tested. Therefore, it is concluded that the drug-loaded stents have great potential for the use in the treatment of intestinal cancers in the future.     

Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo

Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drug-resistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and urea-functionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102 nm with narrow size distribution, were stable in serum-containing solution over 48 h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96 h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. In vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis.     

Spinal cord organotypic slice cultures for the study of regenerating motor axon interactions with 3D scaffolds

Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7–9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon–substrate interactions. By 7–14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.     

Enhanced infarct stabilization and neovascularization mediated by VEGF-loaded PEGylated fibrinogen hydrogel in a rodent myocardial infarction model

Most tissue engineering therapies require biomaterials that are able to induce an angiogenic response to support tissue regeneration. In addition angiogenic growth factor signaling plays an essential role in controlling the process of angiogenesis and matrices have the potential of regulating the concentration of growth factors within the cellular microenvironment. Here we demonstrated myocardial protection and improved post-infarct vascularization of the infarcted hearts using a biosynthetic injectable hydrogel consisting of polyethylene glycol and fibrinogen (PEG-fibrinogen) loaded with vascular endothelial growth factor-A (VEGF-A). Our data revealed PEG-fibrinogen hydrogel was able to store and release VEGF-A in a sustained and controlled fashion. Upon injection after coronary artery ligation, the VEGF-loaded hydrogel significantly improved arteriogenesis and cardiac performance at 4 weeks post-infarction. The results support the future application of PEG-fibrinogen for regulating growth factor signaling in cellular microenvironment and may demonstrates a new strategy for cardiovascular repair with potential for future clinical applications.     

Intracellular redox-activated anticancer drug delivery by functionalized hollow mesoporous silica nanoreservoirs with tumor specificity

In this study, a type of intracellular redox-triggered hollow mesoporous silica nanoreservoirs (HMSNs) with tumor specificity was developed in order to deliver anticancer drug (i.e., doxorubicin (DOX)) to the target tumor cells with high therapeutic efficiency and reduced side effects. Firstly, adamantanamine was grafted onto the orifices of HMSNs using a redox-cleavable disulfide bond as an intermediate linker. Subsequently, a synthetic functional molecule, lactobionic acid-grafted-β-cyclodextrin (β-CD-LA), was immobilized on the surface of HMSNs through specific complexation with the adamantyl group, where β-CD served as an end-capper to keep the loaded drug within HMSNs. β-CD-LA on HMSNs could also act as a targeting agent towards tumor cells (i.e., HepG2 cells), since the lactose group in β-CD-LA is a specific ligand binding with the asialoglycoprotein receptor (ASGP-R) on HepG2 cells. In vitro studies demonstrated that DOX-loaded nanoreservoirs could be selectively endocytosed by HepG2 cells, releasing therapeutic DOX into cytoplasm and efficiently inducing the apoptosis and cell death. In vivo investigations further confirmed that DOX-loaded nanoreservoirs could permeate into the tumor sites and actively interact with tumor cells, which inhibited the tumor growth with the minimized side effect. On the whole, this drug delivery system exhibits a great potential as an efficient carrier for targeted tumor therapy in vitro and in vivo.     

Poly(N-isopropylacrylamide-co-acrylic acid) nanogels for tracing and delivering genes to human mesenchymal stem cells

Drugs, proteins, and cells can be macro- and micro-encapsulated by unique materials that respond to specific stimuli. The phases and hydrophobic interactions of these materials are reversibly altered by environmental stimuli such as pH and temperature. These changes can lead to self-assembly of the materials, which enables controlled drug release and safe gene delivery into cells and tissues. The fate of stem cells delivered by such methods is of great interest. The formation of transgenic tissues requires genes to be delivered safely into stem cells. A cell tracing vehicle and a gene delivery carrier were simultaneously introduced into human mesenchymal stem cells (hMSCs). A thermo-sensitive hydrogel, poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)), was created to generate self-assembled nanoparticles with nanogel characteristics. Hydrophobic interactions mediated the binding of the carboxyl group on the outside of p(NiPAAm-co-AAc) with the amine group of iron oxide. Nanogels carrying iron oxide and a fluorescent dye were complexed with specific genes. These nanogels could be internalized by hMSCs, and the transplantation of these cells into mice was monitored by in vivo imaging. Self-assembled p(NiPAAm-co-dAAc) nanogels complexed with green fluorescent protein were highly expressed in hMSCs and are a potential material for gene delivery.     

Hematopoiesis toxicity induced by CdTe quantum dots determined in an invertebrate model organism

Quantum dots (QDs) have gained significant attention due to their superior optical properties and wide usage in biological and biomedical studies. In recent years, there has been intense concern regarding the in vivo toxicity of QDs. This study was undertaken to examine the toxicity of CdTe QDs on hematopoiesis in an invertebrate model organism, Bombyx mori. Vascular injection of sub-lethal doses of QDs in B. mori larvae caused time- and dose-dependent damage in the hematopoietic organ and hematocytes. QDs with the maximum emission wavelength of 530 nm (QDs530) were quickly observed in cystocytes and plasmacytes, and gradually bleached their green fluorescence, followed by a decrease in peripheral hematocytes. Additionally, the proportion of abnormal hematocytes increased. In marked contrast, QDs with the maximum emission wavelength of 720 nm (QDs720) were quickly surrounded by hematocytes and subsequently enriched in cystocytes like the human's leukocytes, but with weaker cytotoxicity. QDs exposure promoted the mitotic nucleus in prohemocytes and hematocytes similar to peripheral blood stem cells in humans, but aggravated apoptosis. A decrease in hematopoiesis was accompanied by shrinkage and death of hematopoietic organs via an increase in reactive oxygen species. QDs with smaller size resulted in more severe hematopoiesis toxicity.     

The performance of a small-calibre graft for vascular reconstructions in a senescent sheep model

There is an acute clinical need for small-calibre (<6 mm) vascular grafts for surgery. The aim of this study was to evaluate the long-term performance of a small-calibre graft produced from a nanocomposite biomaterial, polyhedral oligomeric silsesquioxane poly(carbonate-urea)urethane (POSS-PCU), in a large animal model following Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP) protocols. Grafts were characterised and implanted into the left carotid artery (LCA) of senescent sheep (n = 11) for a period of 9 months. In vivo compliance and blood flow rates were measured using ultrasound wall tracking software and a Transonic flow meter. Graft patency and degree of intimal hyperplasia (IH) were examined at the study end point. Seven of the POSS-PCU grafts were free from thrombosis, IH, calcification and aneurysmal dilation, with 4 occluding within 14 days. All of the ePTFE controls (n = 4) were found to be occluded by day 32. The lumen of the patent POSS-PCU grafts was free from any cellular deposits, whilst perigraft tissue could be seen to be infiltrating into the body of the graft from the adventitia. No significant differences were detected between the blood flow rates (p = 0.3693) and compliance (p = 0.9706) of the POSS-PCU grafts and the native artery, either post-operatively or after 9 months implantation. Small-calibre vascular grafts produced from POSS-PCU offer a viable option for the clinical use in revascularisation procedures with a patency rate of 64%.     

Mechanisms underlying toxicity induced by CdTe quantum dots determined in an invertebrate model organism

A systematic and thorough quantitative analysis of the in vivo effects of inorganic nanoparticles is extremely important for the design of functional nanomaterials for diagnostic and therapeutic applications, better understanding of their non-specificity toward tissues and cell types, and for assessments of their toxicity. This study was undertaken to examine the impact of CdTe quantum dots (QDs) on an invertebrate freshwater model organism, Hydra vulgaris, for assessment of long term toxicity effects. The continuous exposure of living polyps to sub-lethal doses of QDs caused time and dose dependent morphological damages more severe than Cd²⁺ ions at the same concentrations, impaired both reproductive and regenerative capability, activated biochemical and molecular responses. Of remarkable interest, low QD doses, apparently not effective, caused early changes in the expression of general stress responsive and apoptotic genes. The occurrence of subtle genetic variations, in the absence of morphological damages, indicates the importance of genotoxicity studies for nanoparticle risk assessment. The versatility in morphological, cellular, biochemical and molecular responses renders Hydra a perfect model system for high-throughput screening of toxicological and ecotoxicological impact of nanomaterials on human and environmental health.     

Long term performance of polycaprolactone vascular grafts in a rat abdominal aorta replacement model

In the active field of vascular graft research, polycaprolactone is often used because of its good mechanical strength and its biocompatibility. It is easily processed into micro and nano-fibers by electrospinning to form a porous, cell-friendly scaffold. However, long term in vivo performance of polycaprolactone vascular grafts had yet to be investigated. In this study, polycaprolactone micro and nano-fiber based vascular grafts were evaluated in the rat abdominal aorta replacement model for 1.5, 3, 6, 12, and 18 months (n = 3 for each time point). The grafts were evaluated for patency, thrombosis, compliance, tissue regeneration, and material degradation. Results show excellent structural integrity throughout the study, with no aneurysmal dilation, and perfect patency with no thrombosis and limited intimal hyperplasia. Endothelialization, cell invasion, and neovascularization of the graft wall rapidly increased until 6 months, but at 12 and 18 months, a cellular regression is observed. On the medium term, chondroid metaplasia takes place in the intimal hyperplasia layers, which contributes to calcification of the grafts. This study presents issues with degradable vascular grafts that cannot be identified with short implantation times or in vitro studies. Such findings should allow for better design of next generation vascular grafts.     

The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma

Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction.     

Molecular and structural patterns of bone regeneration in surgically created defects containing bone substitutes

Several biomaterials have been introduced for bone augmentation. However, information is lacking about the mechanisms of bone regeneration and/or integration of these materials in the recipient bone. This study aimed to determine the molecular and structural events in bone defects after augmentation with synthetic tetrapod-shaped calcium phosphate (Tetrabone; TetraB) compared with natural deproteinized bovine bone (DBB). Defects were created in the epiphyses of rat femurs and filled with TetraB or DBB or left empty (Sham). After 3, 6, 14 and 28 d, samples were harvested for histology, histomorphometry, ultrastructure and gene expression analyses. At 3 d, higher expressions of bone formation (ALP and OC) and remodeling (CatK) genes were detected in TetraB compared with DBB and Sham. Downregulation of bone remodeling genes (TRAP and CatK) was detected in DBB as compared to Sham after 14 d. Histomorphometry at 6 and 14 d demonstrated greater bone contact with the granules in TetraB. At 28 d, a larger bone area per defect was found in TetraB. The present experiments show that a synthetic substitute, consisting of α-tricalcium and octacalcium phosphates, induces early osteogenic and osteoclastic activities and promotes bone formation in trabecular bone defects.     

Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation

Endothelial-targeted delivery of antioxidant enzymes, catalase and superoxide dismutase (SOD), is a promising strategy for protecting organs and tissues from inflammation and oxidative stress. Here we describe Protective Antioxidant Carriers for Endothelial Targeting (PACkET), the first carriers capable of targeted endothelial delivery of both catalase and SOD. PACkET formed through controlled precipitation loaded ∼30% enzyme and protected it from proteolytic degradation, whereas attachment of PECAM monoclonal antibodies to surface of the enzyme-loaded carriers, achieved without adversely affecting their stability and functionality, provided targeting. Isotope tracing and microscopy showed that PACkET exhibited specific endothelial binding and internalization in vitro. Endothelial targeting of PACkET was validated in vivo by specific (vs IgG-control) accumulation in the pulmonary vasculature after intravenous injection achieving 33% of injected dose at 30 min. Catalase loaded PACkET protects endothelial cells from killing by H₂O₂ and alleviated the pulmonary edema and leukocyte infiltration in mouse model of endotoxin-induced lung injury, whereas SOD-loaded PACkET mitigated cytokine-induced endothelial pro-inflammatory activation and endotoxin-induced lung inflammation. These studies indicate that PACkET offers a modular approach for vascular targeting of therapeutic enzymes.