Biomineralized hydroxyapatite nanoclay composite scaffolds with polycaprolactone for stem cell-based bone tissue engineering.

Nanoclay modified with unnatural amino acid was used to design a nanoclay-hydroxyapatite (HAP) hybrid by mineralizing HAP in the nanoclay galleries mimicking biomineralization. This hybrid (in situ HAPclay) was used to fabricate polycaprolactone (PCL)/in situ HAPclay films and scaffolds for bone regeneration. Cell culture assays and imaging were used to study interactions between human mesenchymal stem cells (hMSCs) and PCL/in situ HAPclay composites (films and scaffolds). SEM imaging indicated MSC attachment, formation of mineralized extracellular (ECM) on PCL/in situ HAPclay films, and infiltration of MSCs to the interior of PCL/in situ HAPclay scaffolds. Mineralized ECM was formed by MSCs without use of osteogenic supplements. AFM imaging performed on this in vitro generated mineralized ECM on PCL/in situ HAPclay films revealed presence of components (collagen and mineral) of hierarchical organization reminiscent of natural bone. Cellular events observed during two-stage seeding experiments on PCL/in situ HAPclay films indicated similarities with events occurring during in vivo bone formation. PCL/in situ HAPclay films showed significantly increased (100-595% increase in elastic moduli) nanomechanical properties and PCL/in situ HAPclay scaffolds showed increased degradation. This work puts forth PCL/in situ HAPclay composites as viable biomaterials for bone tissue engineering.

Molecular interactions in biomineralized hydroxyapatite amino acid modified nanoclay: in silico design of bone biomaterials.

A simulations driven approach to design of a novel biomaterial nanocomposite system is described in this study. Nanoclays modified with amino acids (OMMT) were used to mineralize hydroxyapatite (HAP), mimicking biomineralization. Representative models of organically modified montmorillonite clay (OMMT) and OMMT-hydroxyapatite (OMMT-HAP) were constructed using molecular dynamics and validated using X-ray Diffraction (XRD), Fourier Transforms Infrared (FTIR) spectroscopy and Transmission Electron Microscopy (TEM). Attractive interactions exist between Ca atoms of HAP and C=O group of aminovaleric acid, indicating chelate formation in OMMT-HAP. Interaction energy maps describe molecular interactions among different constituents and their quantitative contributions in the OMMT and OMMT-HAP systems at both parallel and perpendicular orientations. High attractive and high repulsive interactions were found between PO4(3-) and MMT clay as well as aminovaleric molecules in OMMT-HAP perpendicular and parallel models. Large non-bonded interactions in OMMT-HAP indicate influence of neighboring environment on PO4(3-) in in situ HAPclay. Extensive hydrogen bonds were observed between functional hydrogen atoms of modifier and MMT clay in OMMT-HAP as compared to OMMT. Thus, HAP interacts with clay through the aminovaleric acid. This computational study provides a framework for materials design and selection for biomaterials used in tissue engineering and other areas of regenerative medicine.

pH-triggered echogenicity and contents release from liposomes.

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.

Mmp-9 responsive PEG cleavable nanovesicles for efficient delivery of chemotherapeutics to pancreatic cancer.

Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles' lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional "tumor-like" spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.

Polymer-coated echogenic lipid nanoparticles with dual release triggers.

Although lipid nanoparticles are promising drug delivery vehicles, passive release of encapsulated contents at the target site is often slow. Herein, we report contents release from targeted, polymer-coated, echogenic lipid nanoparticles in the cell cytoplasm by redox trigger and simultaneously enhanced by diagnostic frequency ultrasound. The lipid nanoparticles were polymerized on the external leaflet using a disulfide cross-linker. In the presence of cytosolic concentrations of glutathione, the lipid nanoparticles released 76% of encapsulated contents. Plasma concentrations of glutathione failed to release the encapsulated contents. Application of 3 MHz ultrasound for 2 min simultaneously with the reducing agent enhanced the release to 96%. Folic acid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressing the folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be used as multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Nanoclays mediate stem cell differentiation and mineralized ECM formation on biopolymer scaffolds.

In this work, novel modified nanoclays were used to mineralize hydroxyapatite (HAP) mimicking biomineralization in bone. This in situ HAPclay was further incorporated into chitosan/polygalacturonic acid (Chi/PgA) scaffolds and films for bone tissue engineering. Differences in microstructure of the scaffolds were observed depending on the changes in processing of in situ HAPclay with ChiPgA biopolymer system. Response of human mesenchymal stem cells (hMSCs) on these scaffolds and films was studied using imaging and assays. SEM micrographs indicate that hMSCs were able to adhere to ChiPgA/in situ HAPclay scaffolds and phase contrast images indicated formation of mineralized nodules on ChiPgA/in situ HAPclay films in absence of osteogenic supplements used for differentiation of hMSCs. The formation of mineralized nodules by hMSCs was confirmed by positive staining of the nodules by Alizarin Red S dye. Viability and differentiation assays showed that ChiPgA/in situ HAPclay scaffolds were favorable for viability and differentiation of hMSCs. Unique two-stage cell seeding experiments were performed as a strategy to enhance tissue formation by hMSCs on ChiPgA/in situ HAPclay composite films. This work showed that biomaterials based on ChiPgA/in situ HAPclay composites can be used for bone tissue engineering applications and in situ nanoclay-HAP system mediates osteoinductive and osteoconductive response from hMSCs.

In vitro measurement of attenuation and nonlinear scattering from echogenic liposomes.

Echogenic liposomes (ELIP) are an excellent candidate for concurrent imaging and drug delivery applications. They combine the advantages of liposomes-biocompatibility and ability to encapsulate both hydrophobic and hydrophilic drugs-with strong reflections of ultrasound. The objective of this study is to perform a detailed in vitro acoustic characterization - including nonlinear scattering that has not been studied before - along with an investigation of the primary mechanism of echogenicity. Both components are critical for developing viable clinical applications of ELIP. Mannitol, a cryoprotectant, added during the preparation of ELIP is commonly believed to be critical in making them echogenic. Accordingly, here ELIP prepared with varying amount of mannitol concentration are investigated for their pressure dependent linear and non-linear scattered responses. The average diameter of these liposomes is measured to be 125-185nm. But they have a broad size distribution including liposomes with diameters over a micro-meter as observed by TEM and AFM. These larger liposomes are critical for the overall echogenicity. Attenuation through liposomal solution is measured with four different transducers (central frequencies 2.25, 3.5, 5, 10MHz). Measured attenuation increases linearly with liposome concentration indicating absence of acoustic interactions between liposomes. Due to the broad size distribution, the attenuation shows a flat response without a distinct peak in the range of frequencies (1-12MHz) investigated. A 15-20dB enhancement with 1.67 µg/ml of lipids is observed both for the scattered fundamental and the second harmonic responses at 3.5MHz excitation frequency and 50-800kPa amplitude. It demonstrates the efficacy of ELIP for fundamental as well as harmonic ultrasound imaging. The scattered response however does not show any distinct subharmonic peak for the acoustic excitation parameters studied. Small amount of mannitol proves critical for echogenicity. However, mannitol concentration above 100mM shows no effect.

Use of unnatural amino acids for design of novel organomodified clays as components of nanocomposite biomaterials.

Sodium montmorillonite (Na-MMT) clay was modified with three different unnatural amino acids in order to design intercalated clay structures that may be used for bone biomaterials applications. Prior work on polymer-clay nanocomposites (PCNs) has indicated the effect of the appropriate choice of modifiers on enhancing properties of PCNs. Our X-ray diffraction results indicate an increase in the d-spacing of Na-MMT clay after it was modified with the three unnatural amino acids. Transmission Fourier transform infrared spectroscopy experiments were carried out on the unmodified and modified MMT clay samples to study the molecular interactions between the amino acids used as modifiers and the Na-MMT clay. Cell culture experiments showed that the Na-MMT clay modified with the three amino acids was biocompatible as were the modified clay-incorporated films of chitosan/polygalacturonic acid/hydroxyapatite.