Pt(II)-PLGA Hybrid in a pH-Responsive Nanoparticle System Targeting Ovarian Cancer.

Platinum-based agents are the main treatment option in ovarian cancer (OC). Herein, we report a poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) encapsulating platinum (II), which is targeted to a cell-spanning protein overexpressed in above 90% of late-stage OC, mucin 1 (MUC1). The NP is coated with phospholipid-DNA aptamers against MUC1 and a pH-sensitive PEG derivative containing an acid-labile hydrazone linkage. The pH-sensitive PEG serves as an off-on switch that provides shielding effects at the physiological pH and is shed at lower pH, thus exposing the MUC1 ligands. The pH-MUC1-Pt NPs are stable in the serum and display pH-dependent PEG cleavage and drug release. Moreover, the NPs effectively internalize in OC cells with higher accumulation at lower pH. The Pt (II) loading into the NP was accomplished via PLGA-Pt (II) coordination chemistry and was found to be 1.62 wt.%. In vitro screening using a panel of OC cell lines revealed that pH-MUC1-Pt NP has a greater effect in reducing cellular viability than carboplatin, a clinically relevant drug analogue. Biodistribution studies have demonstrated NP accumulation at tumor sites with effective Pt (II) delivery. Together, these results demonstrate a potential for pH-MUC1-Pt NP for the enhanced Pt (II) therapy of OC and other solid tumors currently treated with platinum agents.

Engineering and Validation of a Peptide-Stabilized Poly(lactic-co-glycolic) Acid Nanoparticle for Targeted Delivery of a Vascular Disruptive Agent in Cancer Therapy.

Developing a biocompatible and biodegradable nanoparticle (NP) carrier that integrates drug-loading capability, active targeting, and imaging modality is extremely challenging. Herein, we report an NP with a core of poly(lactic-co-glycolic) acid (PLGA) chemically modified with the drug combretastatin A4 (CA4), a vascular disrupting agent (VDA) in clinical development for ovarian cancer (OvCA) therapy. The NP is stabilized with a short arginine-glycine-aspartic acid-phenylalanine x3 (RGDFFF) peptide via self-assembly of the peptide on the PLGA surface. Importantly, the use of our RGDFFF coating replaces the commonly used polyethylene glycol (PEG) polymer that itself often induces an unwanted immunogenic response. In addition, the RGD motif of the peptide is well-known to preferentially bind to αvß3 integrin that is implicated in tumor angiogenesis and is exploited as the NP's targeting component. The NP is enhanced with an optical imaging fluorophore label via chemical modification of the PLGA. The RGDFFF-CA4 NPs are synthesized using a nanoprecipitation method and are ∼75 ± 3.7 nm in diameter, where a peptide coating comprises a 2-3 nm outer layer. The NPs are serum stable for 72 h. In vitro studies using human umbilical cord vascular endothelial cells (HUVEC) confirmed the high uptake and biological activity of the RGDFFF-CA4 NP. NP uptake and viability reduction were demonstrated in OvCA cells grown in culture, and the NPs efficiently accumulated in tumors in a preclinical OvCA mouse model. The RGDFFF NP did not induce an inflammatory response when cultured with immune cells. Finally, the NP was efficiently taken up by patient-derived OvCA cells, suggesting a potential for future clinical applications.

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 ± 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

Tripeptide-Stabilized Oil-in-Water Nanoemulsion of an Oleic Acids-Platinum(II) Conjugate as an Anticancer Nanomedicine.

We report a nanoemulsion (NE) which is stabilized by self-assembling tripeptide lysine-tyrosine-phenylalanine (KYF) and encapsulates an oleic acids-platinum conjugate formed using simple Pt (II) coordination chemistry. The KYF-Pt-NE is evaluated both in cultured ovarian cancer cells and in an in vivo preclinical cancer model and shows pH dependent Pt (II) release, which is low at physiological pH and enhanced at tumoral pH. The biological activity of KYF-Pt-NE, evaluated in multiple ovarian cancer cell lines, is significantly higher when compared to the analogous Pt (II) complex used in the clinic. Concurrently, the KYF-Pt-NE platform shows good compatibility with the immune system. Preliminary in vivo testing of KYF-Pt-NE with tumor bearing mice indicates efficient Pt (II) delivery to the tumor. Together, these results demonstrate the potential of peptide-stabilized nanoemulsions, specifically KYF-Pt-NE as an effective nanomedicine against cancer.

Platinum (II) complex-nuclear localization sequence peptide hybrid for overcoming platinum resistance in cancer therapy.

Platinum therapy represents first line of treatment in many malignancies but its high systemic toxicity limits the therapeutic dosage. Herein, we report the synthesis of carboplatin-like complexes with azide and alkyne functional groups and the formation of a platinum (II) - nuclear localization sequence peptide (Pt-NLS) hybrid to improve the import of platinum (II) complexes directly into the cell's nucleus. The Pt-NLS hybrid successfully enters cells and their nuclei, forming Pt-induced nuclear lesions. The in vitro efficacy of Pt-NLS is high, superior to native carboplatin at the same concentration. The methodology used is simple and cost-effective and most importantly can easily be extended to load the Pt (II) onto other supports, opening new possibilities for enhanced delivery of Pt (II) therapy.

A statin-loaded reconstituted high-density lipoprotein nanoparticle inhibits atherosclerotic plaque inflammation.

Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show that this effect is mediated through the inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show that they accumulate in atherosclerotic lesions in which they directly affect plaque macrophages. Finally, we demonstrate that a 3-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a 1-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.

Probing nanoparticle translocation across the permeable endothelium in experimental atherosclerosis.

Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid-polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.

Gold nanocrystal labeling allows low-density lipoprotein imaging from the subcellular to macroscopic level.

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

Synthesis of polymer-lipid nanoparticles for image-guided delivery of dual modality therapy.

For advanced treatment of diseases such as cancer, multicomponent, multifunctional nanoparticles hold great promise. In the current study we report the synthesis of a complex nanoparticle (NP) system with dual drug loading as well as diagnostic properties. To that aim we present a methodology where chemically modified poly(lactic-co-glycolic) acid (PLGA) polymer is formulated into a polymer-lipid NP that contains a cytotoxic drug doxorubicin (DOX) in the polymeric core and an anti-angiogenic drug sorafenib (SRF) in the lipidic corona. The NP core also contains gold nanocrystals (AuNCs) for imaging purposes and cyclodextrin molecules to maximize the DOX encapsulation in the NP core. In addition, a near-infrared (NIR) Cy7 dye was incorporated in the coating. To fabricate the NP we used a microfluidics-based technique that offers unique NP synthesis conditions, which allowed for encapsulation and fine-tuning of optimal ratios of all the NP components. NP phantoms could be visualized with computed tomography (CT) and near-infrared (NIR) fluorescence imaging. We observed timed release of the encapsulated drugs, with fast release of the corona drug SRF and delayed release of a core drug DOX. In tumor bearing mice intravenously administered NPs were found to accumulate at the tumor site by fluorescence imaging.

Synthesis and in vitro evaluation of a multifunctional and surface-switchable nanoemulsion platform.

We present a multifunctional nanoparticle platform that has targeting moieties shielded by a matrix metalloproteinase-2 (MMP2) cleavable PEG coating. Upon incubation with MMP2 this surface-switchable coating is removed and the targeting ligands become available for binding. The concept was evaluated in vitro using biotin and αvß3-integrin-specific RGD-peptide functionalized nanoparticles.

Multifunctional gold nanoparticles for diagnosis and therapy of disease.

Gold nanoparticles (AuNPs) have a number of physical properties that make them appealing for medical applications. For example, the attenuation of X-rays by gold nanoparticles has led to their use in computed tomography imaging and as adjuvants for radiotherapy. AuNPs have numerous other applications in imaging, therapy and diagnostic systems. The advanced state of synthetic chemistry of gold nanoparticles offers precise control over physicochemical and optical properties. Furthermore gold cores are inert and are considered to be biocompatible and nontoxic. The surface of gold nanoparticles can easily be modified for a specific application, and ligands for targeting, drugs or biocompatible coatings can be introduced. AuNPs can be incorporated into larger structures such as polymeric nanoparticles or liposomes that deliver large payloads for enhanced diagnostic applications, efficiently encapsulate drugs for concurrent therapy or add additional imaging labels. This array of features has led to the aforementioned applications in biomedical fields, but more recently in approaches where multifunctional gold nanoparticles are used for multiple methods, such as concurrent diagnosis and therapy, so-called theranostics. This review covers basic principles and recent findings in gold nanoparticle applications for imaging, therapy and diagnostics, with a focus on reports of multifunctional AuNPs.

Inorganic nanocrystals as contrast agents in MRI: synthesis, coating and introduction of multifunctionality.

Inorganic nanocrystals have myriad applications in medicine, including their use as drug or gene delivery complexes, therapeutic hyperthermia agents, in diagnostic systems and as contrast agents in a wide range of medical imaging techniques. In MRI, nanocrystals can produce contrast themselves, with iron oxides having been the most extensively explored, or can be given a coating that generates MR contrast, for example gold nanoparticles coated with gadolinium chelates. These MR-active nanocrystals can be used for imaging of the vasculature, liver and other organs, as well as molecular imaging, cell tracking and theranostics. As a result of these exciting applications, the synthesis and rendering of these nanocrystals as water soluble and biocompatible are therefore highly desirable. We discuss aqueous phase and organic phase methods for the synthesis of inorganic nanocrystals, such as gold, iron oxides and quantum dots. The pros and cons of the various methods are highlighted. We explore various methods for making nanocrystals biocompatible, i.e. direct synthesis of nanocrystals coated with biocompatible coatings, ligand substitution, amphiphile coating and embedding in carrier matrices that can be made biocompatible. Various examples are highlighted and their applications explained. These examples signify that the synthesis of biocompatible nanocrystals with controlled properties has been achieved by numerous research groups and can be applied to a wide range of applications. Therefore, we expect to see reports of preclinical applications of ever more complex MRI-active nanoparticles and their wider exploitation, as well as in novel clinical settings.

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides.

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine-glycine-aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.

Engineering of lipid-coated PLGA nanoparticles with a tunable payload of diagnostically active nanocrystals for medical imaging.

Polylactic-co-glycolic acid (PLGA) based nanoparticles are biocompatible and biodegradable and therefore have been extensively investigated as therapeutic carriers. Here, we engineered diagnostically active PLGA nanoparticles that incorporate high payloads of nanocrystals into their core for tunable bioimaging features. We accomplished this through esterification reactions of PLGA to generate polymers modified with nanocrystals. The PLGA nanoparticles formed from modified PLGA polymers that were functionalized with either gold nanocrystals or quantum dots exhibited favorable features for computed tomography and optical imaging, respectively.

Nanomedical Theranostics in Cardiovascular Disease.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. New diagnostic and therapeutic strategies are needed to mitigate this public health issue. Advances in nanotechnology have generated innovative strategies for diagnosis and therapy in a variety of diseases, foremost in cancer. Based on these studies, a novel concept referred to as nanomedical theranostics, or the combinatory application of nanoparticulate agents to allow diagnostic therapy, is being explored to enable image-guided, personalized, or targeted treatment. Preclinically, theranostics have been gradually applied to CVD with several interesting and encouraging findings. This article summarizes studies and challenges of nanotheranostic strategies in CVD. It also evaluates nanotheranostic strategies that may potentially be utilized to benefit patients.

Silk-silica composites from genetically engineered chimeric proteins: materials properties correlate with silica condensation rate and colloidal stability of the proteins in aqueous solution.

The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL; A1: SGSKGSKRRIL; and Si4-1: MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15-mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](15)X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains (denoted X above), and the results were compared with their non-chimeric counterparts and the silk from Bombyx mori. The effect of pH (3-9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. For the silica-silk-chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction, and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields.

Clay enriched silk biomaterials for bone formation.

The formation of silk protein/clay composite biomaterials for bone tissue formation is described. Silk fibroin serves as an organic scaffolding material offering mechanical stability suitable for bone-specific uses. Clay montmorillonite (Cloisite® Na(+)) and sodium silicate are sources of osteoinductive silica-rich inorganic species, analogous to bioactive bioglass-like bone repair biomaterial systems. Different clay particle-silk composite biomaterial films were compared with silk films doped with sodium silicate as controls for the support of human bone marrow derived mesenchymal stem cells in osteogenic culture. The cells adhered to and proliferated on the silk/clay composites over 2 weeks. Quantitative real time polymerase chain reaction analysis revealed increased transcript levels for alkaline phosphatase, bone sialoprotein, and collagen type 1 osteogenic markers in the cells cultured on the silk/clay films in comparison with the controls. Early evidence of bone formation based on collagen deposition at the cell-biomaterial interface was also found, with more collagen observed for the silk films with higher contents of clay particles. The data suggest that silk/clay composite systems may be useful for further study for bone regenerative needs.

Nanoscale control of silica particle formation via silk-silica fusion proteins for bone regeneration.

The biomimetic design of silk/silica fusion proteins was carried out, combining the self assembling domains of spider dragline silk (Nephila clavipes) and silaffin derived R5 peptide of Cylindrotheca fusiformis that is responsible for silica mineralization. Genetic engineering was used to generate the protein-based biomaterials incorporating the physical properties of both components. With genetic control over the nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, controlled mineralized silk films with different silica morphologies and distributions were successfully generated; generating 3D porous networks, clustered silica nanoparticles (SNPs), or single SNPs. Silk serves as the organic scaffolding to control the material stability and multiprocessing makes silk/silica biomaterials suitable for different tissue regenerative applications. The influence of these new silk-silica composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on the silk/silica films. The presence of the silica in the silk films influenced osteogenic gene expression, with the upregulation of alkaline phosphatase (ALP), bone sialoprotein (BSP), and collagen type 1 (Col 1) markers. Evidence for early bone formation as calcium deposits was observed on silk films with silica. These results indicate the potential utility of these new silk/silica systems towards bone regeneration.

Osteoinductive silk-silica composite biomaterials for bone regeneration.

Osteoinductive and biodegradable composite biomaterials for bone regeneration were prepared by combining silk fibroin with silica particles. The influence of these composite systems on osteogenesis was evaluated with human mesenchymal stem cells (hMSCs) subjected to osteogenic differentiation. hMSCs adhered, proliferated, and differentiated towards osteogenic lineages on silk/silica films. The addition of the silica to the silk films influenced gene expression leading to upregulation of bone sialoprotein (BSP) and collagen type 1 (Col 1) osteogenic markers. Evidence for early bone formation in the form of collagen fibers and apatite nodules was obtained on the silk/silica films. Collagen fibers were closely associated with apatite deposits and overall collagen content was higher for the silica containing samples. Also, smaller sized silica particles (24 nm-2 µm) with large surface area facilitated silica biodegradation in vitro through particle dissolution, leading to ∼5-fold decrease in silica content over 10 weeks. These results indicate the suitability of silk/silica composite system towards bone regeneration, where degradation/remodeling rates of the organic and inorganic components can be controlled.

Smart biomaterials - regulating cell behavior through signaling molecules.

Important advances in the field of tissue engineering are arising from increased interest in novel biomaterial designs with bioactive components that directly influence cell behavior. Following the recent work of Mitchell and co-workers published in BMC Biology, we review how spatial and temporal control of signaling molecules in a matrix material regulates cellular responses for tissue-specific applications.