Analytical performance of a fast multi-element method for titanium and trace elements determination in cosmetics and pharmaceuticals by ICP-AES

A multi-element analytical method based on inductively coupled plasma atomic emission spectrometry (ICP-AES) was developed for trace elements in pharmaceutical tablets and cosmetics. Titanium was also included in the analytes since it is widely used in pharmaceuticals. Critical ICP conditions, like RF incident power, argon gas flow rate and nebulizer sample uptake flow rate were optimized. The most sensitive spectral line of each analyte was selected as optimum for further study. Detection limits in the low μg g⁻¹ range were obtained. Prior to chemical analysis, the samples were decomposed by acid digestion, using various mixtures of HCl, HNO₃ and HF. Yttrium was used as a suitable internal standard in order to correct for possible matrix effects. The method was applied to the analysis of six different pharmaceutical products (anti-biotic, anti-inflammatory, anti-hypertensive) in the form of tablets with film coating and also three cosmetic products like hair and face masks.     

The role of surface science in bioengineered materials

Materials employed in biomedical technology are increasingly being designed to have specific, desirable biological interactions with their surroundings, rather than the older common practice of trying to adapt traditional materials to biomedical applications. Moreover, materials scientists are also increasingly deriving new lessons from naturally occurring materials (from mollusk shells to soft animal tissue) about useful composition–structure property relationships that might be mimicked with synthetic materials. Together, these two areas of effort constitute what we may call bioengineered materials. It is possible to set down a reasonably thorough set of characteristics that bioengineered materials have in common. Among these characteristics we discuss the following: self-assembly, bioengineered materials often rely on information content built into structural molecules to determine the order and organization of the material; hierarchical structure, in most bioengineered materials several different length scales of structure are essential and are formed spontaneously and simultaneously via self-assembly; precision synthesis, fundamental to biological material structures is the idea of macromolecules constructed in a precise manner; templating, ordered structures in bioengineered materials are often propagated from one element or set of instructions, to another; specific and non-specific interactions, the forces involved in holding biomaterials structures together. In the future, a carefully selected combination of this set of characteristics will enable us to bioengineer surfaces that are capable to direct and control a desired biological response. Eventually, such bioengineered surfaces will become important tools to comprehend and analyze how materials interact in nature.     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

PEGylated liposomal doxorubicin targeted to α5β1-expressing MDA-MB-231 breast cancer cells

Targeting drugs selectively to cancer cells can potentially benefit cancer patients by avoiding side effects generally associated with several cancer therapies. One of the attractive approaches to direct the drug cargo to specific sites is to incorporate ligands at the surface of the delivery systems. Integrin α(5)β(1) is overexpressed in tumor vasculature and cancer cells, thus making it an attractive target for use in drug delivery. Our group has developed a fibronectin-mimetic peptide, PR_b, which has been shown to bind specifically to integrin α(5)β(1), thereby providing a tool to target α(5)β(1)-expressing cancer cells in vitro as well as in vivo. Our current work focuses on designing modified stealth liposomes (liposomes functionalized with polyethylene glycol, PEG) for combining the benefits associated with PEGylation, as well as imparting specific targeting properties to the liposomes. We have designed PEGylated liposomes that incorporate in their bilayer the fibronectin-mimetic peptide-amphiphile PR_b that can target several cancer cells that overexpress α(5)β(1), including the MDA-MB-231 breast cancer cells used in this study. We have encapsulated doxorubicin inside the liposomes to enhance its therapeutic potential via PEGylation as well as active targeting to the cancer cells. Our results show that PR_b-functionalized stealth liposomes were able to specifically bind to MDA-MB-231 cells, and the binding could be controlled by varying the peptide concentration. The intracellular trafficking of the doxorubicin liposomes was examined, and within minutes after delivery the majority of them were found to be in the early endosomes, whereas after a longer period of time they had accumulated in the late endosomes and lysosomes. The functionalized liposomes were found to be equally cytotoxic as the free doxorubicin, especially at higher doxorubicin concentrations, and provided higher cytotoxicity than the nontargeted and GRGDSP-functionalized stealth liposomes. Thus, the PR_b-functionalized PEGylated nanoparticles examined in this study offer a promising strategy to deliver their therapeutic payload directly to the breast cancer cells, in an efficient and specific manner.     

Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

Collective and single-molecule interactions of alpha5beta1 integrins

A novel biomimetic system was used to study collective and single-molecule interactions of the alpha5beta1 receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes, which display peptides that mimic the cell adhesion domain of the extracellular matrix protein fibronectin, are constructed from peptide-amphiphiles. The interaction measured with the immobilized alpha5beta1 integrins and GRGDSP peptide-amphiphiles is specifically related to the integrin-peptide binding. It is affected by divalent cations in a way that accurately mimics the adhesion function of the alpha5beta1 receptor. The recognition of the immobilized receptor was significantly increased for a surface that presented both the primary recognition site (GRGDSP) and the synergy site (PHSRN) compared to the adhesion measured with surfaces that displayed only the GRGDSP peptide. At the collective level, the separation process of the receptor-ligand pairs is a combination of multiple unbinding and stretching events that can accurately be described by the wormlike chain (WLC) model of polymer elasticity. In contrast, stretching was not observed at the single-molecule level. The dissociation of single alpha5beta1-GRGDSP pairs under loading rates of 1-305 nN/s revealed the presence of two activation energy barriers in the unbinding process. The high-strength regime above 59 nN/s maps the inner barrier at a distance of 0.09 nm along the direction of the force. Below 59 nN/s a low-strength regime appears with an outer barrier at 2.77 nm and a much slower transition rate that defines the dissociation rate (off-rate) in the absence of force (k(off) degrees = 0.015 s(-1)).     

Interaction Forces between Hydrophobic and Hydrophilic Self-Assembled Monolayers

An investigation is presented of the interaction of charged self-assembled monolayers (SAMs) with a monoprotic ionizable acid functional group (-COOH) and uncharged SAMs with a methyl terminated functional group (-CH(3)). The strength of the interactions are determined using an atomic force microscope. For all electrolyte conditions investigated the interactions are not well described by a summation of van der Waals attractions and electrostatic repulsions in a manner suggesting that van der Waals attractions are screened. The repulsions are accurately described as corresponding to two surfaces of different charge interacting with surface charges that are independent of separation (i.e., the constant charge model). A small adhesion force was observed under all conditions and its magnitude increased with NaCl concentration. Copyright 2000 Academic Press.     

Design of a novel fibronectin-mimetic peptide-amphiphile for functionalized biomaterials

The interaction of the alpha5beta1 integrin with its ligand, fibronectin, supports numerous adhesive functions and has an important role in health and disease. In recent years, there has been a considerable effort in designing fibronectin-mimetic peptides to target the integrin. However, to date, the therapeutic use of these peptides has been limited, as they cannot accurately mimic fibronectin's binding affinity for alpha5beta1. A peptide-amphiphile (PR_b) was synthesized with a peptide headgroup composed of four building blocks: a spacer; RGDSP, the primary recognition site for alpha5beta1; PHSRN, the synergy binding site; and a linker. The linker was designed to mimic two important criteria: the distance and the hydrophobicity/hydrophilicity between PHSRN and RGD in fibronectin. Human umbilical vein endothelial cells were seeded on different substrates and evaluated in terms of adhesion, spreading, specificity, cytoskeleton organization, focal adhesions, and secretion of extracellular fibronectin. This peptide was shown to perform comparably to fibronectin, indicating that a biomimetic approach can result in the design of novel peptides with therapeutic potential for biomaterial functionalization.     

Patterned biomimetic membranes: effect of concentration and pH

Planar-supported lipid bilayers have attracted enormous attention because of their properties as model cell membranes, which can be employed in a variety of fundamental biological studies and medical devices. Furthermore, the development of patterned biological interfaces is of great practical and scientific interest because of their potential applications in the field of biosensors, drug screening, tissue engineering, and medical implants. In this study, mica-supported membranes were constructed from biomimetic peptide-amphiphiles and their mixtures with lipidated poly(ethylene glycol) (PEG120) molecules or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids using the Langmuir-Blodgett technique. The two peptide-amphiphiles used in this study were a fibronectin-mimetic with the PHSRN(SG)(3)SGRGDSP headgroup (referred to as PHSRN-GRGDSP) that contains both the primary (GRGDSP) and the synergy (PHSRN) recognition sites for alpha(5)beta(1) integrins and a peptide-amphiphile that mimics a fragment of the N-terminus of the fractalkine receptor (referred to as NTFR). Compression isotherms of the peptide-amphiphiles and their mixtures with PEG120 at the air/water interface were recorded and analyzed to evaluate the extent of miscibility in the two-component LB films. Domain formation in mica-supported bilayers constructed from mixtures of peptide-amphiphiles and lipidated PEG120 or DPPC was observed using atomic force microscopy. In PHSRN-GRGDSP/PEG120 mixtures deposited from an aqueous subphase at pH 7, concentration-dependent phase separation was observed on the AFM images. The NTFR/PEG120 and NTFR/DPPC mixtures deposited at pH 10 exhibited extensive lateral phase separation at all mixture compositions, whereas at deposition pH 7 the concentrations of NTFR/DPPC examined here were well mixed.