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GFP expression levels from MB-435 tumor tissue sections at different levels ( Â 40 objective). Section A which was close to the SELP+virus injection site had higher GFP expression than section C. Section C was approximately 4 Y 5 mm apart from section A. GFP expression was localized to the cells in proximity of the injection site.
Source publication
The purpose of this study was to investigate the potential of silk-elastinlike protein polymers (SELPs) in controlling the release rate of adenoviruses in vitro and in vivo while preserving their bioactivity.
A hydrogel system composed of SELP/adenovirus mixture was prepared. The release of the adenovirus particles from the hydrogels was quantified...
Context in source publication
Context 1
... changing polymer concentration it is possible to control virus delivery up to 28 days. The 4% gel with 50 ul size released 100% of the loaded viruses in one week, followed by more than 70% release from 6% gel, and up to 20% from 8% v/v hydrogels (Fig. 2). The release profiles of adenoviruses from 4, 6 and 8% gels were significantly different ( p <0.05) from each other. An increase in polymer concentration leads to increased hydrogel crosslinking density resulting in decreased rate of virus release. To determine the infectivity of the released viruses, SELP discs (100 m l) containing Ad.GFP were incubated in complete growth media. At predetermined time points samples were drawn to measure the number of released viruses (Fig. 3a) and the corresponding green fluorescence activity as a qualitative measure of virus infectivity (Fig. 3b). Results demonstrate that SELP hydrogels can sustain the release of the adenoviral particles over the 28 day period of study and preserve their infectivity. After a series of experiments in various release media (i.e., PBS, DMEM, DMEM/ Serum), different gel sizes (i.e., 50 m l and 100 m l) and number of loaded viruses (10 8 Y 10 9 ), we obtained sustained release of infectious adenoviruses over the period of 28 days. The bioactivity of the released adenoviruses was also measured quantitatively. SELP discs containing Ad.CMV.LacZ were incubated in complete growth media and the number of released viruses was measured at each time point (Fig. 4a). Under the same conditions but in a separate set of samples, at each time point samples were withdrawn and used to transduce HeLa cells followed by the b -gal activity measurements [Fig. 4b(a)]. The b -gal activity of the released viruses from the gels [Fig. 4b(a)] was compared with the b -gal activity of the same number of viruses with 100% infectivity [Fig. 4b(b)] ( n =3, p <0.05). It was observed that the decline in transduction efficiency of the viruses is due to the combination of decrease in release rate and virus inactivation during incubation in complete growth media over the 28 days. The results also show 30 Y 40% loss of virus activity in this period. It is noteworthy that in our studies, after the incubation of the viruses (no SELP) in the complete growth media in the presence of serum, virus infectivity was detectable only for 9 T 3 days (Mean T S.E., n =9). The preservation of virus infectivity in the SELP matrix could be the result of the stabilization of the virus envelop by the silk or elastin units, although this needs to be explored more in depth. To examine the ability of SELP matrices in localizing and prolonging viral transduction in vivo , murine models of breast (MDA-MB-435) and head and neck (Fadu) tumor xenografts were used. The animal protocol in this study has been approved by the University of Maryland Institutional Animal Care and Use Committee. The control group received 4 Â 10 9 plaque forming units of Ad.GFP per tumor while the test group received 50 m l of SELP mixed with 4 Â 10 9 plaque forming units. Our previous studies have shown that SELP by itself does not have any green fluorescent activity (21). The results of the MB-435 and Fadu control group injected with viruses (no SELP), revealed that the tumors infected with Ad.GFP could express GFP up to 15 days with expression levels decreasing on days 11 and 15. The significant reduction in GFP expression at later days could be the result of the virus inactivation by the mouse immune system, leakage of viruses to the blood stream and clearance by liver, or combination thereof. In the first week of the study, the distribution of GFP expression in MB-435 tumors was uniform at all levels of the tumor sections suggesting the free convection of viruses throughout the tumor tissue whereas patchy in Fadu tumors. This could be related to the existing barriers within Fadu tumors preventing uniform viral spread. Overall, the level of GFP expression in MB-435 tumors was much higher than Fadu tumors which could be due to the higher expression level of CAR (Coxsackie and Adenovirus Receptor) on the surface of MB-435 tumor cells. The number of adenovirus receptor on the surface of the tumor cells can play a significant role in the outcome of the adenoviral cancer gene therapy (22,23). The test group infected with the mixture of SELP+virus showed persistent GFP expression up to 15 days confirming the compatibility of SELP with adenoviruses in preserving their activity (Fig. 5). Overall, the GFP expression level up to day 15 was much higher in SELP+virus group than virus only, suggesting the possible utility of these polymers for localized and sustained adenoviral delivery. Although in both control and test groups high transduction levels were observed up to day 11, in test group (SELP+virus) at day 15 GFP expression was mostly observed in the proximity of the injection site and minimum to no apparent cell transduction in the tumor tissue farther from the injection site (Fig. 6). This could be the result of virus entrapment in the gel matrix leading to reduced rate of dissemination to distant sites within tumors. This finding is important as it suggests SELP is capable of entrapping viruses and of forming a virus depot to localize its effect. These results are in agreement with previously reported studies (12) and corroborate the potential of virus laden hydrogel systems as an alternative to direct intratumoral virus ...
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Citations
... Hydrogels could also keep the infectivity of the cargo intact. Of interest, SELP can be designed to be temperature sensitive [114]. Minimally invasive injection of the liquid polymer in situ showed that the firm and irreversible hydrogel was formed when stimulated by body temperature, later releasing the encapsulated therapeutics upon gradual degradation of the matrix. ...
Hydrogel scaffolds hold great promise for developing novel treatment strategies in the field of regenerative medicine. Within this context, silk fibroin (SF) has proven to be a versatile material for a wide range of tissue engineering applications owing to its structural and functional properties. In the present review, we report on the design and fabrication of different forms of SF-based scaffolds for tissue regeneration applications, particularly for skin, bone, and neural tissues. In particular, SF hydrogels have emerged as delivery systems for a wide range of bio-actives. Given the growing interest in the field, this review has a primary focus on the fabrication, characterization, and properties of SF hydrogels. We also discuss their potential for the delivery of drugs, stem cells, genes, peptides, and growth factors, including future directions in the field of SF hydrogel scaffolds.
... In a breast cancer-bearing mouse model, SELP hydrogel was used to deliver Renilla luciferase plasmid in vivo which resulted in a substantial improvement in luciferase gene expression and the preservation of transfection potency, which was the better option inside the tumor. Hatefi et al. [131] looked into adenoviral delivery using SELPs in vitro and in vivo. The stabilization virus interaction with silk or elastin units was due to the sustained release in vitro of imprisoned adenoviruses with the retained operation of infection after 28 days. ...
Silk fibroin (SF), an organic material obtained from the cocoons of a silkworm Bombyx mori, is used in several applications and has a proven track record in biomedicine owing to its superior compatibility with the human body, superb mechanical characteristics, and its controllable propensity to decay. Due to its robust biocompatibility, less immunogenic, non-toxic, non-carcinogenic, and biodegradable properties, it has been widely used in biological and biomedical fields, including wound healing. The key strategies for building diverse SF-based drug delivery systems are discussed in this review, as well as the most recent ways for developing functionalized SF for controlled or redirected medicines, gene therapy, and wound healing. Understanding the features of SF and the various ways to manipulate its physicochemical and mechanical properties enables the development of more effective drug delivery devices. Drugs are encapsulated in SF-based drug delivery systems to extend their shelf life and control their release, allowing them to travel further across the bloodstream and thus extend their range of operation. Furthermore, due to their tunable properties, SF-based drug delivery systems open up new possibilities for drug delivery, gene therapy, and wound healing.
... Silk-elastins are artificial proteins that were developed by genetic engineering and biological production methods using Escherichia coli [7]. Silk-elastins have a unique feature of temperature-mediated self-gelation [8], which has led to the development of biomaterials using silk-elastin: delivery systems for drugs and viruses [9,10], 3-dimensional tissue-engineering scaffolds [11], and wound healing materials [12][13][14][15]. We have previously shown with in vitro studies and animal models that SE-P47K, a type of silk-elastin, promotes wound healing [13][14][15][16]. ...
IntroductionNot only chronic but also some acute wounds have a risk of infection and become unhealed wounds. Silk-elastin sponge has been developed to treat chronic wounds that are susceptible to infection. Preclinical and clinical studies suggested that silk-elastin sponge is safe for humans and can promote granulation tissue formation by reducing bacterial growth in chronic wounds. The central aim of this trial is to evaluate the clinical utility and safety of silk-elastin sponge for the treatment of chronic and acute skin ulcers.Methods
This study is a prospective, multicenter, single-arm, uncontrolled clinical trial. In this study, 20 patients with chronic ulcers and five with an acute one will be included; patients with wound infection will be excluded. Silk-elastin sponges are applied and covered with a dressing for 14 days.Planned OutcomesThe primary endpoint is the frequency of patients with chronic wounds in whom the investigator confirms the formation of a healthy wound bed at 14 days after the initial application of the study device. In addition, safety for acute wounds and handiness of the study device will be assessed.Trial registration numberjRCT2052210072.
... Hatefi et al loaded SELP-47K, an SELP comprised of four SLP and 7 ELP blocks with one ELP block containing a Lys substitution, with the adenoviruses Ad.GFP or Ad.CMV.LacZ that encode green fluorescent protein (GFP) or β-galactosidase, respectively [405]. In an in vitro study, the SELP hydrogel maintained the release of the adenoviruses over a 28 day period with only a 30-40% loss in virus activity ( Figure 24A) [405]. ...
... Hatefi et al loaded SELP-47K, an SELP comprised of four SLP and 7 ELP blocks with one ELP block containing a Lys substitution, with the adenoviruses Ad.GFP or Ad.CMV.LacZ that encode green fluorescent protein (GFP) or β-galactosidase, respectively [405]. In an in vitro study, the SELP hydrogel maintained the release of the adenoviruses over a 28 day period with only a 30-40% loss in virus activity ( Figure 24A) [405]. The Ad.GFP-loaded SELP was intratumorally showed significantly lower GFP expression than tumors receiving the SELP formulation ( Figure 24B) [405]. ...
... In an in vitro study, the SELP hydrogel maintained the release of the adenoviruses over a 28 day period with only a 30-40% loss in virus activity ( Figure 24A) [405]. The Ad.GFP-loaded SELP was intratumorally showed significantly lower GFP expression than tumors receiving the SELP formulation ( Figure 24B) [405]. This study demonstrated the potential of SELPs for adenoviral delivery to solid tumors. ...
Engineering protein and peptide-based materials for drug delivery applications has gained momentum due to their biochemical and biophysical properties over synthetic materials, including biocompatibility, ease of synthesis and purification, tunability, scalability, and lack of toxicity. These biomolecules have been used to develop a host of drug delivery platforms, such as peptide- and protein-drug conjugates, injectable particles, and drug depots to deliver small molecule drugs, therapeutic proteins, and nucleic acids. In this review, we discuss progress in engineering the architecture and biological functions of peptide-based biomaterials -naturally derived, chemically synthesized and recombinant- with a focus on the molecular features that modulate their structure-function relationships for drug delivery.
... Zeng et al. showed that microscale porous hydrogels are derived from interconnected clusters of polymer nanofibers ( Figure 6). [134] The stimuli-responsive behavior of SELPs and the diversity of structures they form, combined with post-processing techniques such as electrospinning or thin film deposition, [135,136] have made them useful for various applications in drug delivery (chemoembolics, [137] in situ enemas, [138] and adenovirus delivery [139] ) as well as in tissue engineering, [140] and wound healing. [141] ...
Well‐defined tunable nanostructures formed through the hierarchical self‐assembly of peptide building blocks have drawn significant attention due to their potential applications in biomedical science. Artificial protein polymers derived from elastin‐like polypeptides (ELPs), which are based on the repeating sequence of tropoelastin (the water‐soluble precursor to elastin), provide a promising platform for creating nanostructures due to their biocompatibility, ease of synthesis, and customizable architecture. By designing the sequence and composition of ELPs at the gene level, their physicochemical properties can be controlled to a degree that is unmatched by synthetic polymers. A variety of ELP‐based nanostructures are designed, inspired by the self‐assembly of elastin and other proteins in biological systems. The choice of building blocks determines not only the physical properties of the nanostructures, but also their self‐assembly into architectures ranging from spherical micelles to elongated nanofibers. This review focuses on the molecular determinants of ELP and ELP‐hybrid self‐assembly and formation of spherical, rod‐like, worm‐like, fibrillar, and vesicle architectures. A brief discussion of the potential biomedical applications of these supramolecular assemblies is also included.
... The in vivo intratumoral delivery of Renilla luciferase plasmid using SELP hydrogel in a breast cancer-bearing mouse model exhibited a significant increase in the gene expression of luciferase and retention of transfection efficacy, which was preferable within the tumor. Hatefi et al. further analyzed the in vitro and in vivo adenoviral delivery using SELPs [141]. The in vitro sustained release of entrapped adenoviruses with maintained infection activity was observed within 28 days and was attributed to the stabilization effect between the silk or elastin units and virus. ...
Since it was first discovered, thousands of years ago, silkworm silk has been known to be an abundant biopolymer with a vast range of attractive properties. The utilization of silk fibroin (SF), the main protein of silkworm silk, has not been limited to the textile industry but has been further extended to various high-tech application areas, including biomaterials for drug delivery systems and tissue engineering. The outstanding mechanical properties of SF, including its facile processability, superior biocompatibility, controllable biodegradation, and versatile functionalization have allowed its use for innovative applications. In this review, we describe the structure, composition, general properties, and structure-properties relationship of SF. In addition, the methods used for the fabrication and modification of various materials are briefly addressed. Lastly, recent applications of SF-based materials for small molecule drug delivery, biological drug delivery, gene therapy, wound healing, and bone regeneration are reviewed and our perspectives on future development of these favorable materials are also shared.
... 48 To visualize viral replication in tumor tissues, immunohistochemistry was conducted with Ad-2/5 E1A-specific antibody (SC-430; Santa Cruz Biotechnology, USA) as described elsewhere. 49 ...
A dual pH- and temperature-responsive physically crosslinked and injectable hydrogel system was developed for efficient and long-term delivery of oncolytic adenoviruses (Ads). Three different type of physically crosslinked hydrogels with different chemical compositions and properties were prepared. These hydrogels with good biocompatibility can be injected at pH 9.0 and room temperature and rapidly form a gel at body or tumor microenvironment condition. Ads encapsulated in hydrogels was released gradually without burst release. Moreover, these physically crosslinked hydrogels provided a protective environment for Ads and retained their bioactivity for a long period of time. Compared to naked Ads, Ads protected by these physically crosslinked hydrogels showed strong cytotoxicity to cancer cells even after 11 days. Ads-loaded hydrogel system also exhibited enhanced and long-term antitumor therapeutic effects in human xenograft tumor models. Due to these outstanding properties, Ads-loaded injectable hydrogel might have potential for long-term cancer treatment.
... Further, a histological study revealed that more abundant Ad particles were present three weeks following virus administration in tumor those treated with Ad encapsulated in SELP hydrogel in comparison to tissues treated with naked Ad. These results are in a good agreement with previous studies in which intratumorally administered naked Ad was shown to rapidly disseminate into the surrounding tissue and cleared out by host immunity [109,111,112], resulting in poor viral accumulation in tumor tissues. Ad-mediated suppression of C-Met expression in tumor tissue was also significantly enhanced and prolonged by encapsulation with SELP matrix. ...
Viral vectors are promising gene carriers for cancer therapy. However, virus-mediated gene therapies have demonstrated insufficient therapeutic efficacy in clinical trials due to rapid dissemination to nontarget tissues and to the immunogenicity of viral vectors, resulting in poor retention at the disease locus and induction of adverse inflammatory responses in patients. Further, the limited tropism of viral vectors prevents efficient gene delivery to target tissues. In this regard, modification of the viral surface with nanomaterials is a promising strategy to augment vector accumulation at the target tissue, circumvent the host immune response, and avoid nonspecific interactions with the reticuloendothelial system or serum complement. In the present review, we discuss various chemical modification strategies to enhance the therapeutic efficacy of viral vectors delivered either locally or systemically. We conclude by highlighting the salient features of various nanomaterial-coated viral vectors and their prospects and directions for future research.
... Collagen (Chandler et al., 2000;Cohen-Sacks et al., 2004;Doukas et al., 2002;Doukas et al., 2001;Hijjawi et al., 2004;McMahon et al., 2011;Schek et al., 2004; Shin and Shea, 2010) and silk-elastin-like protein (SELP) biomaterials (Cresce et al., 2008;Greish et al., 2010;Gustafson et al., 2009;Gustafson et al., 2010;Hatefi et al., 2007;Megeed et al., 2004;Price et al., 2012;Price et al., 2015) have been further manipulated to generate systems for the delivery of nonviral (CohenSacks et al., 2004;Doukas et al., 2002;Hijjawi et al., 2004;Megeed et al., 2004) and viral (lentiviral and adenoviral) vectors (Chandler et al., 2000;Cresce et al., 2008;Doukas et al., 2001;Greish et al., 2010;Gustafson et al., 2009;Gustafson et al., 2010;Hatefi et al., 2007;McMahon et al., 2011;Price et al., 2012;Price et al., 2015;Schek et al., 2004;) in skin, bone, muscle, brain, and blood vessels and to treat ischemia and tumors. Self-assembling peptide hydrogels (ReyRico et al., 2015a;Zheng et al., 2015) are also valuable systems that have been employed thus far for cartilage repair upon gene transfer of lentiviral (Zheng et al., 2015) and rAAV vectors (ReyRico et al., 2015a).Adenoviral Bone, wound, vessels (Breen et al., 2006;Schek et al., 2004;Wan et al., 2006) rAAV Cartilage (Lee et al., 2011) Gelatin Nonviral Muscle, diabetes, ischemia (Aoyama et al., 2003;Fukunaka et al., 2002;Kasahara et al., 2003;Kushibiki et al., 2005;Kushibiki et al., 2003) Adenoviral Tumor (Okino et al., 2003) Alginate Adenoviral Tumor (Wang et al., 2003) rAAV Cartilage (Diaz-Rodriguez et al., 2015)Collagen Nonviral Muscle, vessels, ischemia (Cohen-Sacks et al., 2004;Doukas et al., 2002;Hijjawi et al., 2004) Lentiviral Wound, brain (McMahon et al., 2011;Adenoviral Wound, vessels, bone (Chandler et al., 2000;Doukas et al., 2001;Schek et al., 2004)SELP Nonviral Tumor (Megeed et al., 2004) Adenoviral Tumor (Cresce et al., 2008;Greish et al., 2010;Gustafson et al., 2009;Gustafson et al., 2010;Hatefi et al., 2007;Price et al., 2012;Price et al., 2015) Self-assembling peptides Lentiviral Cartilage (Zheng et al., 2015)rAAV Cartilage (Rey-Rico et al., 2015a) Pluronic Nonviral Controlled release (Chun et al., 2005) Lentiviral Brain (Strappe et al., 2005) Adenoviral Tumor (Wang et al., 2005)rAAV Tumor, cartilage (Driessens et al., 2011;Rey-Rico et al., 2015b)OPF Nonviral Controlled release (Kasper et al., 2005)PLGA Nonviral Vessels, wound, skin (Eliaz & Szoka, 2002;Lee et al., 2003;) Adenoviral Tumor (Davidson et al., 1997)Abbreviations: SELP, silk-elastin-like protein; OPF, oligo(poly(ethylene glycol) fumarate; PLGA, poly(lactic-glycolic) acid. nDiscovery Medicine, Volume 21, Number 11x, 2016Novel Gene Delivery Systems for Gene Therapy ...
Even though gene therapy made its way through the clinics to treat a number of human pathologies since the early years of experimental research and despite the recent approval of the first gene-based product (Glybera) in Europe, the safe and effective use of gene transfer vectors remains a challenge in human gene therapy due to the existence of barriers in the host organism. While work is under active investigation to improve the gene transfer systems themselves, the use of controlled release approaches may offer alternative, convenient tools of vector delivery to achieve a performant gene transfer in vivo while overcoming the various physiological barriers that preclude its wide use in patients. This article provides an overview of the most significant contributions showing how the principles of controlled release strategies may be adapted for human gene therapy.
... Since these studies, different parameters have been examined, such as SELP and DNA concentration, SELP silk-elastin ratio and molecular weight, SELP hydrogel cure time, which influence the release of DNA and adenovirus in vitro and in vivo. For DNA as well as for virus delivery, the release was inversely correlated to concentration of SELPs and SELP hydrogel cure time [114,[116][117][118]. Increased concentration of the polymer led to an increase of the density prevented loss of embedded DNA or virus. ...
Introduction:
Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semi-crystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli-responsive properties and tunability, thereby becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release.
Areas covered:
An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli-responsive features and current and potential drug delivery applications.
Expert opinion:
The tunable material properties allow SELPs to be pursued as promising biomaterials for nanocarriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli-responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration.
