Designed proteinoid polymers and nanoparticles encapsulating risperidone for enhanced antipsychotic activity.

BACKGROUND: Nanoparticles (NPs) incorporating drug formulations can be used to facilitate passage through biological barriers including the blood-brain barrier (BBB) and increase drug delivery and bioavailability. Hence, NP-based administration may enhance the efficiency of current antipsychotics. Encapsulation within NPs can resolve aqueous solubility problems that not only reduce permeability through the BBB but also affect targeting. The present study describes a new drug delivery system based on proteinoid NPs to explore the possibility of improving drug efficacy. Risperidone (RSP) is a commonly used atypical antipsychotic medication, and was therefore selected for encapsulation by proteinoid NPs. RESULTS: Proteinoid polymers with high molecular weight and low polydispersity were synthesized from L-amino acids and poly-L-lactic acid (PLLA) by thermal step-growth polymerization mechanism. RSP-loaded proteinoid NPs were then prepared using a self-assembly process in the presence of RSP, followed by PEGylation. The optimal PEGylated RSP-loaded NPs were characterized in terms of diameter and size distribution, drug loading, ζ-potential, cytotoxicity, biodistribution, and psychopharmacological effects. The findings indicate significantly higher antipsychotic activity of drug-loaded proteinoid NPs compared to free RSP. CONCLUSIONS: Proteinoid NPs enhance RSP delivery and may potentially increase drug efficiency by reducing dosage and side effects.

Solidification of oil liquids by encapsulation within porous hollow silica microspheres of narrow size distribution for pharmaceutical and cosmetic applications.

This study presents a new process for hydrophilic formulation of liquid oils, by encapsulation and solidification of the oils within porous hollow silica microspheres of narrow size distribution. Jojoba [Simmondsia chinensis] oil was chosen as a model study due to its broad potential applications. Jojoba oil is produced from the seeds of the jojoba plant, which are rich in liquid wax. Today, jojoba oil is mainly used for applications such as pharmaceuticals and cosmetics. The oil is primarily used as a carrier oil that stabilizes sensitive active compounds, such as vitamins and other oils, which are susceptible to air oxidation or UV-light degradation. Silica (SiO2) particles are used in many different industrial products such as food and cosmetics due to their chemical inertness. Here, uniform porous hollow SiO2 microspheres, composed of sintered SiO2 nanoparticles, were made by coating polystyrene template microspheres of narrow size distribution with three layers of SiO2 nanoparticles, followed by removal of the polystyrene core by combustion at 500 °C. The synthesis stages were characterized by SEM, TEM, FTIR and TGA analyses. The measurements confirmed the increasing content of SiO2 after each coating cycle and the absence of polystyrene in the final hollow particles. Jojoba oil was successfully encapsulated within the hollow SiO2 microspheres by heating/cooling cycles, reaching an encapsulation yield of up to 10 times of the SiO2 dry shell weight. The oil encapsulation was confirmed by a floatability test and confocal microscopy. The hollow SiO2 and the oil-filled microspheres were found non-toxic to HaCaT cell line, a spontaneously transformed human epithelial cell line from adult skin. Furthermore, the oil-filled SiO2 microspheres were dispersed in a hydrogel and exhibited a homogeneous water-based formulation that appeared stable after six months storage. In light of these findings, we offer these jojoba oil-filled particles as a model for hydrophilic formulation of oils in general and in particular as suitable candidates for pharmaceutical and cosmetic applications.

Targeted drug delivery of near IR fluorescent doxorubicin-conjugated poly(ethylene glycol) bisphosphonate nanoparticles for diagnosis and therapy of primary and metastatic bone cancer in a mouse model.

BACKGROUND: Most primary and metastatic bone tumors demonstrate increased osteoclast activity and bone resorption. Current treatment is based on a combination of surgery, radiotherapy and chemotherapy. Severe side effects are associated with chemotherapy due to use of high dosage and nonspecific uptake. Bisphosphonates have a strong affinity to Ca2+ ions and are widely used in the treatment of bone disorders. RESULTS: We have engineered a unique biodegradable bisphosphonate nanoparticle (NPs) bearing two functional surface groups: (1) primary amine groups for covalent attachment of a dye/drug (e.g. NIR dye Cy 7 or doxorubicin); (2) bisphosphonate groups for targeting and chelation to bone hydroxyapatite. In addition, these engineered NPs contain high polyethyleneglycol (PEG) concentration in order to increase their blood half life time. In vitro experiments on Saos-2 human osteosarcoma cell line, demonstrated that at a tenth of the concentration, doxorubicin-conjugated bisphosphonate NPs achieved a similar uptake to free doxorubicin. In vivo targeting experiments using the NIR fluorescence bisphosphonate NPs on both Soas-2 human osteosarcoma xenograft mouse model and orthotopic bone metastases mCherry-labeled 4T1 breast cancer mouse model confirmed specific targeting. In addition, therapeutic in vivo experiments using doxorubicin-conjugated bisphosphonate NPs demonstrated a 40% greater inhibition of tumor growth in Saos-2 human osteosarcoma xenograft mouse model when compared to free doxorubicin. CONCLUSIONS: In this research we have shown the potential use of doxorubicin-conjugated BP NPs for the targeting and treatment of primary and metastatic bone tumors. The targeted delivery of doxorubicin to the tumor significantly increased the efficacy of the anti-cancer drug, thus enabling the effective use of a lower concentration of doxorubicin. Furthermore, the targeting ability of the BP NPs in an orthotopic xenograft mouse model reinforced our findings that these BP NPs have the potential to be used for the treatment of primary and metastatic bone cancer.

Near IR fluorescent conjugated poly(ethylene glycol)bisphosphonate nanoparticles for in vivo bone targeting in a young mouse model.

Bisphosphonate (BP) compounds are widely used in the treatment of bone disorders. This group of drugs with a high affinity to Ca(+2) ions is rapidly attracted to bone mineral, especially in areas of high resorption. We have engineered unique biodegradable BP nanoparticles (NPs) by dispersion co-polymerization of the monomers methacrylate-PEG-BP) and (3-Aminopropyl)mathacrylamide) with the crosslinker monomer tetra ethylene glycol diacrylate. These NPs possess a dual functionality: (1) covalent attachment of a dye (e.g. near IR dye) or a drug to the nanoparticles through the primary amine groups on the surface of the NPs; (2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the NPs. This study describes the uptake of the unique near IR fluorescent Cy 7-conjugated BP NPs in bone of a young mouse model. Blood half-life studies revealed a relatively long half-life (approximately 5 h) due to a high concentration of PEG in the BP NPs as well as a relatively long whole body clearance (approximately 2 weeks). Body distribution studies showed a specific uptake of the BP NPs in bone. These unique engineered BP NPs are planned to be utilized in future work for diagnostic and drug delivery systems that are targeted to bone disorders.

Magnetic micro-device for manipulating PC12 cell migration and organization.

Directing neuronal migration and growth has an important impact on potential post traumatic therapies. Magnetic manipulation is an advantageous method for remotely guiding cells. In the present study, we have generated highly localized magnetic fields with controllable magnetic flux densities to manipulate neuron-like cell migration and organization at the microscale level. We designed and fabricated a unique miniaturized magnetic device composed of an array of rectangular ferromagnetic bars made of permalloy (Ni80Fe20), sputter-deposited onto glass substrates. The asymmetric shape of the magnets enables one to design a magnetic landscape with high flux densities at the poles. Iron oxide nanoparticles were introduced into PC12 cells, making the cells magnetically sensitive. First, we manipulated the cells by applying an external magnetic field. The magnetic force was strong enough to direct PC12 cell migration in culture. Based on time lapse observations, we analysed the movement of the cells and estimated the amount of MNPs per cell. We plated the uploaded cells on the micro-patterned magnetic device. The cells migrated towards the high magnetic flux zones and aggregated at the edges of the patterned magnets, corroborating that the cells with magnetic nanoparticles are indeed affected by the micro-magnets and attracted to the bars' magnetic poles. Our study presents an emerging method for the generation of pre-programmed magnetic micro-'hot spots' to locate and direct cellular growth, setting the stage for implanted magnetic devices.

NGF-conjugated iron oxide nanoparticles promote differentiation and outgrowth of PC12 cells.

The search for regenerative agents that promote neuronal differentiation and repair is of great importance. Nerve growth factor (NGF) which is an essential contributor to neuronal differentiation has shown high pharmacological potential for the treatment of central neurodegenerative diseases such as Alzheimer's and Parkinson's. However, growth factors undergo rapid degradation, leading to a short biological half-life. In our study, we describe a new nano-based approach to enhance the NGF activity resulting in promoted neuronal differentiation. We covalently conjugated NGF to iron oxide nanoparticles (NGF-NPs) and studied the effect of the novel complex on the differentiation of PC12 cells. We found that the NGF-NP treatment, at the same concentration as free NGF, significantly promoted neurite outgrowth and increased the complexity of the neuronal branching trees. Examination of neuronal differentiation gene markers demonstrated higher levels of expression in PC12 cells treated with the conjugated factor. By manipulating the NGF specific receptor, TrkA, we have demonstrated that NGF-NPs induce cell differentiation via the regular pathway. Importantly, we have shown that NGF-NPs undergo slower degradation than free NGF, extending their half-life and increasing NGF availability. Even a low concentration of conjugated NGF treatment has led to an effective response. We propose the use of the NGF-NP complex which has magnetic characteristics, also as a useful method to enhance NGF efficiency and activity, thus, paving the way for substantial neuronal repair therapeutics.

The support-on-support concept for in-situ oligonucleotide synthesis on nanoparticles.

Oligonucleotide-loaded nanoparticles, which are of interest for biomedical application, up to now, could not be prepared by in-situ synthesis, due to difficulty of handling in automated synthesizers. To overcome this problem, we have introduced the "support-on-support" concept. It is based on the reversible anchoring of nanoparticles to the surface of microparticles. These composite beads easily can be used for automated synthesis, being released after completion of chain elongations. As examples, dextran-coated magnetite nanoparticles were attached to polystyrene microparticles through (1) a gelatine or (2) a silica layer. Release involved dissolution of the bonding layer by (1) proteases or (2) alkali.

Glial cell line-derived neurotrophic factor-conjugated nanoparticles suppress acquisition of cocaine self-administration in rats.

The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) may have therapeutic potential for preventing and treating cocaine addiction. Previously, we found that transplantation of a GDNF-expressing astrocyte cell line into the striatum and nucleus accumbens attenuates cocaine-seeking behavior in Sprague-Dawley rats. However, as a potential treatment for humans, cell transplantation presents several technical and ethical complications. Nanoparticulate systems are a safe and effective method for introducing exogenous compounds into the brain. Therefore, we examined the effect of GDNF-conjugated nanoparticles microinjected into the striatum and nucleus accumbens on cocaine self-administration in rats. GDNF-conjugated nanoparticles blocked the acquisition of cocaine self-administration compared to control treatments. Furthermore, a cocaine dose response demonstrated that decreased lever response in rats that received GDNF-conjugated nanoparticles persisted after substitution with different cocaine doses. This effect is not due to a non-specific disruption of locomotor or operant behavior, as seen following a water operant task. The current study is one of the first demonstrations that drug-conjugated nanoparticles may be effective in treating brain disorders. These findings suggest that GDNF-conjugated nanoparticles may serve as a novel potential treatment for drug addiction.

Mobility measurements of immunomagnetically labeled cells allow quantitation of secondary antibody binding amplification.

Magnetic cell separation methods commonly utilize paramagnetic materials conjugated to antibodies that target specific cell surface molecules. The amount of magnetic material bound to a cell is directly proportional to the magnetophoretic mobility of that cell. A mathematical model has been developed which characterizes the fundamental parameters controlling the amount of magnetic material bound, and thus, the magnetophoretic mobility of an immunomagnetically labeled cell. In characterization of the paramagnetic labeling, one of the parameters of interest is the increase in magnetophoretic mobility due to the secondary antibody binding to multiple epitopes on the primary antibody, referred to as the "secondary antibody binding amplification," Psi. Secondary antibody-binding amplification has been investigated and quantitated by comparing the mobilities of lymphocytes directly labeled with anti-CD4 MACS (Miltenyi Biotec, Auburn, CA) magnetic nanoparticle antibody with the mobilities of lymphocytes from the same sample labeled with two different indirect antibody-labeling schemes. Each indirect labeling scheme incorporated a primary mouse anti-CD4 FITC antibody that provides both FITC and mouse-specific binding sites for two different secondary antibody-magnetic nanoparticle conjugates: either anti-FITC MACS magnetic nanoparticle antibody or anti-mouse MACS magnetic nanoparticle antibody. The magnetophoretic mobilities of the immunomagnetically labeled cells were obtained using Cell Tracking Velocimetry (CTV). The results indicate that an average of 3.4 anti-FITC MACS magnetic nanoparticle antibodies bind to each primary CD4 FITC antibody, Psi(1,2f) = 3.4 +/- 0.33, and that approximately one, Psi(1,2m) = 0.98 +/- 0.081, anti-mouse MACS magnetic nanoparticle antibody binds to each primary mouse CD4 FITC antibody on a CD4 positive lymphocyte. These results have provided a better understanding of the antibody-binding mechanisms used in paramagnetic cell labeling for magnetic cell separation.

Polymer support for exonucleolytic sequencing.

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene "core" and a "shell" of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 microm diameter. Applying M13 ssDNA in extremely high dilution (approximately 1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 microm diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand.

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry.

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.

Study of magnetic particles pulse-injected into an annular SPLITT-like channel inside a quadrupole magnetic field.

Advantages of the continuous magnetic flow sorting for biomedical applications over current, batch-wise magnetic separations include high throughput and a potential for scale-up operations. A continuous magnetic sorting process has been developed based on the quadrupole magnetic field centered on an annular flow channel. The performance of the sorter has been described using the conceptual framework of split-flow thin (SPLITT) fractionation, a derivative of field-flow fractionation (FFF). To eliminate the variability inherent in working with a heterogenous cell population, we developed a set of monodisperse magnetic microspheres of a characteristic magnetization, and a magnetophoretic mobility, similar to those of the cells labeled with a magnetic colloid. The theory of the magnetic sorting process has been tested by injecting a suspension of the magnetic beads into the carrier fluid flowing through the sorter and by comparing the theoretical and experimental recovery versus total flow-rate profiles. The position of the recovery maxima along the total flow-rate axis was a function of the average bead magnetophoretic mobility and the magnetic field intensity. The theory has correctly predicted the position of the peak maxima on the total flow-rate axis and the dependence on the bead mobility and the field intensity, but has not correctly predicted the peak heights. The differences between the calculated and the measured peak heights were a function of the total flow-rate through the system, indicating a fluid-mechanical origin of the deviations from the theory (such as expected of the lift force effects in the system). The well-controlled elution studies using the monodisperse magnetic beads, and the SPLITT theory, provided us with a firm basis for the future sorter evaluation using cell mixtures.

Solid-supported oligonucleotide systems for special biomedical applications.

The use of composite beads consisting of a 6 microns polystyrene core with 30 nm surface-bound silica particles to routine automatic oligodeoxynucleotide (ODN) synthesis is described.

Synthesis, characterization, and use of immobilized polyacrolein microspheres in diagnostics: a model determination of alpha 1-antitrypsin in human serum.

Polyacrolein microspheres covalently coupled onto solid surfaces, e.g., glass, silicon crystals, and polystyrene, have been prepared and characterized. These surfaces were synthesized by derivatization of the substrate surfaces with SiCl3(CH2)3CN or Si(OEt)3-(CH2)4NH2. The terminal nitrile groups were then reduced to primary amine groups. Polyacrolein microspheres of various diameters (0.08 and 0.4 microns) were then covalently bound in a monolayer structure to the modified surfaces. This binding of the microspheres to the derivatized surfaces is achieved via polyvalent Schiff-base bonds formed by the interaction between aldehyde groups of the microspheres and omega-primary amine groups of the modified surfaces. Residual amine groups were blocked with acetic acid N-hydroxysuccinimide ester. The residual aldehyde groups of the immobilized microspheres can then be used for covalent binding of amino ligands, e.g., proteins, in a single step and at physiological pH (or any other desired pH). The potential use of the immobilized polyacrolein microsphere surfaces for diagnostics has been demonstrated by determination of alpha 1-antitrypsin in human serum with trypsin bound to the immobilized microspheres. The comparison between this new method for the determination of alpha 1-antitrypsin and the routine methods is discussed.

Body distribution of 75Se-radiolabeled silica nanoparticles covalently coated with omega-functionalized surfactants after intravenous injection in rats.

Silica nanoparticles, radiolabeled with 75Selenium were coated with 14 types of omega-functionalized surfactants covalently bound to the particle surface. The particles were suspended in phosphate buffered saline (PBS) and injected intravenously via the tail vein of Wistar rats. The animals were sacrificed after 5 different time points (30 min, 2 h, 6 h, 24 h, and 7 d), and two samples of each organ and two blood samples were weighed into vials. The radioactivity of each sample was measured in a LKB-Wallac CliniGamma counter. Coated silica nanoparticles accumulated in the liver at much lower levels than other colloidal drug carriers after short time periods (30 min). The liver accumulation increased after longer time periods due to a natural redistribution process. Surface modification by increasing the hydrophilicity and thickness of coating yielded higher and longer persisting concentrations in the intestine, blood, and the muscles. Initially increased lung concentrations were decreasing with time, probably due to migration of the alveolar phagocytes.

Peptide, protein, and cellular interactions with self-assembled monolayer model surfaces.

Model biomaterial surfaces with well defined chemistry were prepared from close-packed alkyltrichlorosilane monolayers on polished silicon and glass. The outermost molecular groups which come in direct contact with the biological environment were varied across a wide range of oxidation states by employing -CF3, -CH3, -CO2CH3, and -CH2OH terminal functionalities. Characterization by contact angles, surface spectroscopy, and ellipsometry verified that these model surfaces could be repeatedly prepared with good consistency for routine use to study biomolecule adsorption onto model surfaces. Adhesion of canine endothelial cells and the adsorption of proteins (human serum albumin and human fibrinogen) as well as series of synthetic defined oligopeptides to these model surfaces have been studied. Endothelial cells attachment and growth were in the rank order of: -CH2OH > -CO2Me > -CH3 > -CF3. The peptides were comprised of different alternating sequences of lysine, leucine, and tryptophan residues. These structural differences imparted different amphiphilic characters that led to measurable differences in the adsorption of these peptides to liquid-vapor interfaces. The adsorption to model surfaces was studied using ESCA, radiometry, and concentration-dependent contact angles. ESCA and radiometry measured irreversible biomolecules adsorption whereas the contact angle method measured steady-state adsorption. Radiometric results were inconsistent with ESCA, possibly due to artifacts associated with protein radiolabeling.

Affinity separation with polyaldehyde microsphere beads.

Agarose polyaldehyde microsphere beads were prepared by encapsulating polyaldehyde microspheres of various diameters, e.g., polyacrolein or polyglutaraldehyde microspheres, within agarose beads. Amino ligands such as proteins or drugs can be bound covalently to the beads in a single step at physiological pH. The binding capacity of the beads towards various amino ligands is inversely related to the diameter of the microspheres encapsulated in the agarose matrix. Different reagents, e.g., bovine serum albumin, ethanolamine and hydroxylamine, were studied as blocking reagents of the free aldehyde groups. Blocking the remaining aldehyde groups after coupling the amino ligands to the beads is essential for increasing or retaining the reactivity of the ligands conjugated to the beads. Among the reagent studied, hydroxylamine was found to be the most suitable blocking reagent of the free aldehyde groups of beads conjugated with proteins. The extent of leakage of amino ligands bound to the agarose-polyaldehyde microsphere beads was studied as a function of the pH of aqueous solutions of the beads. At physiological pH the leakage was negligible. At acid pH, leakage of ligands containing several primary amine groups, e.g., proteins, was insignificant. However, significant leakage was detected for ligands containing a single amino group. The leakage of proteins bound to the agarose-polyaldehyde microsphere beads was found to be much less than the leakage of the same proteins bound to agarose beads through the cyanogen bromide activation method.

Removal of breast cancer cells by soybean agglutinin in an experimental model for purging human marrow.

Soybean agglutinin (SBA) was used as a differential reagent to achieve selective elimination of human breast cancer cells (T-47D cell line) from human marrow contaminated with tumor cells. Two successive cycles of direct agglutination by soluble SBA resulted in depletion of 3.5 logs of tumor cells as determined by radiolabeling, whereas removal of more than 4 logs of tumor cells was demonstrated by a clonogenic bioassay. A more convenient procedure for tumor purge involved the use of SBA bound to either polyglutaraldehyde magnetic beads or to commercial polystyrene magnetic beads. After one cycle of magnetic separation, 2 to 3.5 logs of tumor cells were removed. A second separation cycle using fresh magnetic beads improved depletion to more than 4 logs. Neither of these purging procedures affected the hematopoietic potential of granuloid-macrophage colony-forming unit cells. We suggest the use of SBA bound to magnetic beads as a convenient tool for effective ex vivo purging of marrow aspirates contaminated with metastatic breast cancer cells in patients with advanced disease. A similar procedure is applicable for all SBA-positive neoplasms.

Treatment of adverse digitalis effects by hemoperfusion through columns with antidigoxin antibodies bound to agarose polyacrolein microsphere beads.

Ten patients with an array of moderate to severe adverse effects resulting from digitalis were effectively treated by hemoperfusion through small columns which contained antidigoxin antibodies bound to polyacrolein microspheres in agarose macrospheres (APAMB). The procedure was well tolerated. There was no detectable damage to formed blood elements and no changes in electrolytes, liver enzymes, or other related biochemical parameters. Despite some theoretic considerations to the contrary, the removal of a relatively small load of digoxin resulted in amelioration of the clinical symptoms and ECG abnormalities associated with digitalis. No rebound phenomena of intoxication or posthemoperfusion increase in digoxin serum levels were noted over the subsequent 5 to 6 days. A further increase in the capacity of the columns may render this method a safe and convenient emergency procedure for patients with digitalis toxicity.

Purging breast cancer cells in preparation for autologous bone marrow transplantation.

Mixtures of the T-47D human breast cancer cell line and normal human bone marrow cells were used for studying a new approach for purging epithelial tumor cells for autologous bone marrow transplantation (BMT) in breast cancer. Breast cancer cell line T-47D cells were shown to bind soybean agglutinin (SBA) in a specific fashion that could be blocked by D-galactose. Tumor cells were effectively purged by both SBA agglutination and depletion of cells bound to magnetic beads (0.7-5.0 micron) covalently linked to SBA. A depletion of 3-4 orders of magnitude of tumor cells was consistently accomplished by combining one step of agglutination followed by one cycle of SBA-magnetic bead depletion. Neither procedure affects stem cell recovery. We suggest that effective purging of breast cancer cells can be accomplished using SBA for autologous BMT in patients with advanced breast cancer.