Glycol Spacer Research Articles

Folate‐conjugated β‐cyclodextrin from click chemistry strategy and for tumor‐targeted drug delivery

To enhance site-specific intracellular delivery against the folate receptor, a drug carrier was designed and synthesized by bioconjugation of folic acid (FA) to β-cyclodextrins (β-CD) through a poly(ethylene glycol) (PEG) spacer from "click chemistry" strategy. The resulted conjugates were confirmed by (1)H NMR and IR spectroscopy. Host-guest interactions between hydrophobic drug and β-CD are capable of entrapping a hydrophobic drug, like 5-Fluorouracil, to form drug-β-CD-PEG-FA nanoparticles (NPs) in aqueous solution. The morphology and size of β-CD-PEG-FA NPs were measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The targeting ability of the β-CD-PEG-FA NPs was investigated against two kinds of cell lines (HeLa and A549), which have different amounts of folate receptors on their surface. Confocal image analysis revealed that β-CD-PEG-FA conjugate-assembled nanoparticles exhibited a greater extent of cellular uptake against HeLa cells than A549 cells. This suggests folate-receptor-mediated endocytosis can affect the cellular uptake efficiency of drug-loaded β-CD-PEG-FA NPs. The β-CD-PEG-FA conjugates that are presented may be promising active tumor-targeting carrier candidates via folate mediation.

ELISA and SPR Studies of Ricin Binding to β-Galactoside Analogs

Early detection of ricin toxin, a biothreat agent, is of critical importance because it can be produced in large quantities rapidly. Ricin binds to β-galactoside analogs; therefore, these glycans can be used to capture and detect the toxin. Here, we have compared the binding affinities (Kds) of ricin with a panel of β-galactoside analogs using two different biophysical techniques, quantitative enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). We found that β-galactosides bind to ricin better than N-acetyl β-galactosamines on all surfaces. Second, ricin prefers to bind to densely packed glycans over sparsely packed glycans. Third, when the structure and density of the glycans are kept constant and only the spacer is varied, we found that the binding affinities of glycans attached via a hydrocarbon spacer are better than glycans attached via a short oligoethylene glycol spacer, presumably because the alkyl spacer provides a better spatial arrangement. We also observed that the ELISA platform is more sensitive than the SPR platform. Taken together, in addition to the glycan structure, glycan density, presentation, and assay conditions are important factors that modulate the binding affinities. These comparative quantitative studies revealed similarities and subtle differences in the binding of the same glycans attached to different surfaces and must be taken into consideration when developing glycan-based biosensors for ricin or other glycan binding proteins.

Targeting prostate cancer cells with a multivalent PSMA inhibitor-guided streptavidin conjugate

Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin–biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG4-CTT54, and biotin-PEG12-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC50 values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG12-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG12-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC50=1.86nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA’s biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

BackgroundCell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.MethodsA hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.ResultsThe reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization.ConclusionsThe data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

Highly sensitive detection of protein phosphorylation by using improved Phos‐tag Biotin

We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.

Toll-Like Receptor-7 Agonist Decoration Enhances the Adjuvanticity of Chitosan–DNA Nanoparticles

In order to provide an adjuvant-equipped carrier system for plasmid deoxyribonucleic acid (pDNA) vaccines, we grafted for the first time a Toll-like receptor (TLR)-7 agonistic moiety [9-benzyl-8-hydroxyadenine (HA)] through a poly(ethylene glycol) (PEG) spacer onto a water-soluble chitosan derivative [final copolymer: 6-0-carboxymethyl-N,N,N-trimethylchitosan (CTC)-graft-PEG-HA (CTCPHA)]. Successful grafting was confirmed by spectroscopic (H NMR, mass, ultraviolet-visible, and Fourier transform infrared spectroscopy) and chromatographic (size-exclusion chromatography-multi-angle laser light scattering) methods. In this article, TLR-7 agonist-decorated CTCPHA nanoparticles (NPs) were formulated by complex coacervation with pDNA expressing the green fluorescence protein. Resulting NPs had a size of around 200 nm with a positive surface charge and high DNA encapsulation efficiency. In contrast to the use of DNA alone, NP protected DNA against enzymatic degradation and enabled transfection of alveolar A549 cells. Interestingly, TLR-7 agonist decoration increased significantly the interleukin-8-related immune stimulatory capacity of polymeric chitosan and chitosan-based NP in human THP-1 macrophages when compared with controls. In summary, we demonstrate here the proof-of-principle that covalent TLR-7 agonist functionalization of chitosan-DNA NPs enhances the carrier's adjuvanticity, representing a valuable concept for future polymer-based DNA vaccination.

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3′ and 5′ version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an “on-line” assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.

Syntheses of deuterium‐labelled cholesteryl neoglycolipids

Four deuterium‐labelled neoglycolipids derived from cholesterol were synthesized for embedment into liposomes. Deuterium atoms were either incorporated by CH2 replacement with a CD2 group in the triethylene glycol spacer arm between the cholesteryl residue and the sugar moiety (products 2–4) or incorporated directly on the acetamido function in the sugar head (compound 5).

Factors Affecting the Endothelial Retention of Targeted Microbubbles: Influence of Microbubble Shell Design and Cell Surface Projection of the Endothelial Target Molecule

In biologic systems, the arrest of circulating cells is mediated by adhesion molecules projecting their active binding domain above the cell surface to enhance bond formation and tether strength. Similarly, molecular spacers are used for ligands on particle-based molecular imaging agents. The aim of this study was to evaluate the influence of tether length for targeting ligands on ultrasound molecular imaging agents. Microbubbles bearing biotin at the end of variable-length polyethylene glycol spacer arms (MB(2000) and MB(3400)) were prepared. To assess invivo attachment efficiency to endothelial counterligands that vary in their distance from the endothelial cell surface, contrast-enhanced ultrasound (CEU) molecular imaging of tumor necrosis factor-α-induced P-selectin (long distance) or intercellular adhesion molecule-2 (short distance) was performed with each agent in murine hind limbs. To assess the influence of the glycocalyx on microbubble attachment, CEU molecular imaging of intercellular adhesion molecule-2 was performed after degradation of the glycocalyx. CEU molecular imaging targeted to P-selectin showed signal enhancement above control agent for MB(2000) and MB(3400), the degree of which was significantly higher for MB(3400) compared with MB(2000). CEU molecular imaging targeted to intercellular adhesion molecule-2 showed low overall signal for all agents and signal enhancement above control for MB(3400) only. Glycocalyx degradation increased signal for MB(3400) and MB(2000). Microbubble targeting to endothelial ligands is influenced by (1) the tether length of the ligand, (2)the degree to which the endothelial target is projected from the cell surface, and (3) the status of the glycocalyx. These considerations are important for designing targeted imaging probes and understanding potential obstacles to molecular imaging.

Synthesis and Characterization of Novel Polythiophenes Containing Azobenzene Units and Well-Defined Oligo(Ethylene Glycol) Spacers: Thermal and Optical Properties, and Preparation of Langmuir Films

A new series of 3-alkyl-ester-substituted polythiophenes, bearing amino-nitro substituted azobenzenes and flexible tetra(ethylene glycol) spacers, have been synthesized and characterized. First an amphiphilic monomer (E)-2-(4-((4-nitrophenyl)diazenyl)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl 2-(tiophen-3-yl) acetate (AT) was prepared and homo-polymerized to give the corresponding polymer poly[(E)-2-(4((4-nitrophenyl)diazenyl)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl 2-(tiophen-3-yl) acetate] (HP). This monomer was also co-polymerized in the presence of 3-butylthiophene, 3-hexylthiophene and 3-dodecylthiophene to give the corresponding co-polymers CP4, CP6 and CP12. These polymers exhibited molecular weights ranging between 16 500 and 46 990 g/mol. HP showed lower thermal stability than the corresponding co-polymers CP4, CP6 and CP12. The use of 3-alkylthiophene plastificant co-monomers conferred higher thermal stability to the obtained co-polymers. Polymers bearing higher azobenzene content showed to be more susceptible to degradation. All polymers exhibited a maximum absorption band at λ max = 456–475 nm, due to the amino-nitro-substituted azobenzene units. Finally, Langmuir films were prepared with HP and CP4, whose formation was monitored by plotting their surface pressure vs. molecular area (π/A) isotherms and by Brewster angle microscopy (BAM).

Synthesis and Characterization of Novel Amphiphilic Azo-Polymers Bearing Well-Defined Oligo(Ethylene Glycol) Spacers

Four novel azo-polymers bearing oligo(ethylene glycol) spacers were synthesized and characterized. Monomers were prepared by esterification of the RED-PEG dyes previously synthesized by us: N-methyl-N-{4-[(E)-(4-nitrophenyl)diazenyl]phenyl}-N-(5-hydroxy-3-oxapentas-1-yl)amine (RED-PEG-2), N-methylN-{4-[(E)-(4-nitrophenyl)diazenyl]phenyl}-N-(8-hydroxy-3,6-dioxaoctas-1-yl)amine (RED-PEG-3), N-methyl-N-{4-[(E)-(4-nitrophenyl)diazenyl]phenyl}-N-(11-hydroxy-3,6,9-trioxaundecas-1-yl)amine (RED-PEG-4) and N-methyl-N-{4-[(E)-(4-nitrophenyl)diazenyl]phenyl}-N-(17-hydroxy-3,6,9,12,15-pentaoxaheptadecas-1-yl)amine (RED-PEG-6) with methacryloyl chloride. The obtained monomers (nPEGMAN series) were polymerized in the presence of AIBN in THF to give the corresponding azo-polymers (pnPEGMAN series). Also, Langmuir films were prepared with these polymers; the obtained monolayers exhibited good reversible behavior upon successive compression/expansion cycles.

Design of near-infrared fluorescent bioactive conjugated functional iron oxide nanoparticles for optical detection of colon cancer

BackgroundColon cancer is one of the major causes of death in the Western world. Early detection significantly improves long-term survival for patients with the disease. Near- infrared (NIR) fluorescent nanoparticles hold great promise as contrast agents for tumor detection. NIR offers several advantages for bioimaging compared with fluorescence in the visible spectrum, ie, lower autofluorescence of biological tissues, lower absorbance, and consequently deeper penetration into biomatrices.Methods and resultsNIR fluorescent iron oxide nanoparticles with a narrow size distribution were prepared by nucleation, followed by controlled growth of thin iron oxide films onto cyanine NIR dye conjugated gelatin-iron oxide nuclei. For functionalization, and in order to increase the NIR fluorescence intensity, the NIR fluorescent iron oxide nanoparticles obtained were coated with human serum albumin containing cyanine NIR dye. Leakage of the NIR dye from these nanoparticles into phosphate-buffered saline solution containing 4% albumin was not detected. The work presented here is a feasibility study to test the suitability of iron oxide-human serum albumin NIR fluorescent nanoparticles for optical detection of colon cancer. It demonstrates that encapsulation of NIR fluorescent dye within these nanoparticles significantly reduces photobleaching of the dye. Tumor-targeting ligands, peanut agglutinin and anticarcinoembryonic antigen antibodies (αCEA), were covalently conjugated with the NIR fluorescent iron oxide-human serum albumin nanoparticles via a poly(ethylene glycol) spacer. Specific colon tumor detection was demonstrated in chicken embryo and mouse models for both nonconjugated and the peanut agglutinin-conjugated or αCEA-conjugated NIR fluorescent iron oxide-human serum albumin nanoparticles.ConclusionConjugation of peanut agglutinin or αCEA to the nanoparticles significantly increased the fluorescence intensity of the tagged colon tumor tissues relative to the nonconjugated nanoparticles.

Morphogen-driven self-construction of covalent films built from polyelectrolytes and homobifunctional spacers: buildup and pH response

Self-construction of films using polyelectrolytes bearing complementary chemical groups is a recent emerging concept in the coating field. Herein, we describe the one-pot morphogen film self-construction through formation of covalent bonds between polyelectrolytes and homobifunctional spacers by Huisgen click-chemistry. One-pot solution of poly(acrylic acid) bearing alkyne groups (PAA-Alk), bisazides ethylene glycol (Az-EGn-Az) spacers and CuSO4 was brought into contact with a gold electrode. Cu(I), the morphogen, was generated electrochemically from Cu(II) at the electrode/one-pot solution interface. The PAA-Alk/Az-EGn-Az film buildup was investigated by varying the length of the EGn spacer (n = 2, 13 and 50) leading to different growth rates. By increasing the pH of the solution, a reversible swelling of the films (up to 400% for n = 13 or 50) is obtained due to electrostatic repulsion between PAA chains. Further investigations on film swelling were performed by using PAA-Alk and an original bisazide spacer based on two pyrene units connected by a triamine spacer (Az-bispyrene-Az). This spacer has the property to adopt an extended conformation under acidic conditions and a “stacked” conformation under basic conditions, which both can be monitored by fluorimetry. Despite the mechanical tension due to film swelling at basic pH, bispyrene spacers adopt a stacked conformation. This allowed us to monitor the pH within PAA-Alk/Az-bispyrene-Az films. The pH remains fairly stable and below the pKa of PAA when the pH of the contacting solution varies from pH 5 to pH 7 and increases above pH 8, which explains why film swelling takes place essentially above pH 8.

Synthesis of α-biotinyl poly(ethylene glycol-b-N-isopropylacrylamide) block copolymers with different fluorescent dyes at the ω-side

A series of α-biotinylated poly(N-isopropylacrylamide) (PNIPAAm) block copolymers with different poly(ethylene glycol) (PEG) spacers were synthesized and two approaches for ω-functionalization were tested, first, by the use of atom transfer radical polymerization (ATRP) and sequential monomer addition and second, by halogen end group modification with azides and subsequent copper catalyzed azide–alkyne cycloadditions. Three different dyes, rhodamine B, fluorescein and a coumarin derivative, were chosen as end groups. For each approach the corresponding monomer or alkyne functionalized dye was synthesized. The biotin group can be used for attaching the polymers to streptavidin whereas the fluorescent dyes can be used as labels for polymer monitoring in various environments and thus to larger structures (e.g. microtubule, surfaces, etc.). The synthesis routes of the biotin–PEG initiators were developed in a way that also amino functionalized PEG initiators as intermediates were produced. The advantage of this approach is the use of these polymers for other purposes like direct covalent coupling to biomolecules, fluorescent labeling or surface attachment, gaining a very flexible tool for PNIPAAm functionalization. The two approaches for ω-functionalization were investigated by NMR, GPC and UV-Vis spectroscopy and compared for their efficiency.

Hydrogels with well-defined peptide-hydrogel spacing and concentration: impact on epithelial cell behavior

The spacing of peptides away from a hydrogel matrix dramatically impacts their availability and subsequent interactions with cells. Peptides were synthesized with monodisperse poly(ethylene glycol) spacers of different lengths that separate the peptide from the monomeric functionality which reacts during hydrogel polymerization. Specifically, bioactive RGD ligands were conjugated to PEG(5), PEG(11) or PEG(27) spacers via solid phase techniques and then functionalized with an acryloyl end group. These acryloyl-PEGx-RGD conjugates were then copolymerized with PEGDA to form an inert hydrogel network decorated with RGD ligands for cell interactions. As the PEG spacer length increases, the RGD concentration required to support cell attachment and spreading decreases. The competitive detachment of hTCEpi cells in the presence of soluble linear RGD also shows non-linear dependence on the PEG spacer length, as more cells remained attached and spread on gels functionalized with longer PEG-RGD conjugates in comparison to the shorter PEG-RGD conjugates. The strategy and synthetic techniques developed here allow for reproducible control over peptide-hydrogel spacing and peptide concentration, and may be extended for incorporation of multiple peptides and to other hydrogel platforms.

Impact of precursor size on the chain structure and mechanical properties of solvent-swollen epoxy gels

The thermal and mechanical properties of solvent-swollen epoxy gels were investigated as a function of pre-cursor size in a model end-linked network. The epoxy networks were formed by cross-linking diepoxide and diamine precursors in the presence of a low volatility solvent, dibutylphthalate (DBP). Both precursors were end functionalized and contained a poly(propylene glycol) (PPG) spacer between the epoxy and amine functionality, respectively. The lengths of both precursors were controlled by varying the molecular weight of the PPG spacer between functional groups. The glass transition temperature for the gels as a function of solvent loading was well predicted by the Fox equation. The scaling factors of shear storage modulus versus solvent loading increased with increasing diamine precursor molecular weight to values much larger (3.91) than the theoretical value of 2.3, for entanglement dominated network formation in a theta solvent. In contrast, the scaling factor increased with decreasing epoxy precursor molecular weight to values near 4.5. The large, molecular weight dependent scaling factors are attributed to loop defect formation as the result of the amine cross-linker architecture, which consists of two difunctional reactive end groups separated by a spacer. Rather than equal spacing of all four reactive sites, the amine end groups contain two reactive hydrogens that increase the local concentration of reactive species, and facilitates loop formation. We anticipate that this work will aid in the development of non-aqueous gels and provide enhanced tailoring of the gel properties over a broad range of stiffness.

Peptide-modified liposomes for selective targeting of bombesin receptors overexpressed by cancer cells: a potential theranostic agent

ObjectivesDrug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors.MethodsA new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized.ResultsLiposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with peptide-free DSPC liposomes (1.4% ± 0.2% at 37°C). Incubation of cells with DSPC/ MonY-BN/Dox showed significantly lower cell survival compared with DSPC/Dox-treated cells, in the presence of 100 ng/mL and 300 ng/mL drug amounts, in cytotoxicity experiments. Intravenous treatment of PC-3 xenograft-bearing mice with DSPC/MonY-BN/Dox at 10 mg/kg Dox dose produced higher tumour growth inhibition (60%) compared with nonspecific DSPC/ Dox liposomes (36%) relative to control animals.ConclusionThe structural and loading properties of DSPC/MonY-BN liposomes along with the observed in-vitro and in-vivo activity are encouraging for further development of this approach for target-specific cancer chemotherapy.

Synthesis, Pharmacoscintigraphic Evaluation and Antitumor Efficacy of Methotrexate-Loaded, Folate-Conjugated, Stealth Albumin Nanoparticles

The present study aims to develop a multifunctional nanoformulation based on technetium-99m labeled, folate conjugated, methotrexate-loaded human serum albumin nanoparticles (HSA NPs) and explore their potential in cancer theragnostics. Methotrexate-loaded HSA NPs were synthesized by a reverse microemulsion technique, followed by chemical crosslinking with glutaraldehyde. These NPs were conjugated with folic acid (FA) through a hydrophilic polyethylene glycol spacer to render them long-circulatory and augment their tumor-specific localization. The therapeutic conjugate was further radiolabeled with a γ-emitter technetium-99m for real-time monitoring of its blood clearance kinetics and biodistribution through the measurement of blood/organ-associated radioactivity and scintigraphic imaging. In vitro cell-uptake and cytotoxicity studies corroborated that FA conjugation enabled these NPs to specifically target and kill folate-receptor overexpressing cancer cells via S phase arrest. Blood clearance kinetics and biodistribution studies clearly indicated that circulation time, as well as tumor-specific localization of methotrexate-loaded HSA nanocarriers, could be significantly augmented upon polyethylene glycolylation and conjugation of FA. Finally, we demonstrated that these targeted HSA NPs inhibited tumor growth more effectively, as compared with the nontargeted controls.

Innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA

A tumor targeted mesoporous silica nanoparticles (MSN)-based drug delivery system (DDS) was developed for inhalation treatment of lung cancer. The system was capable of effectively delivering inside cancer cells anticancer drugs (doxorubicin and cisplatin) combined with two types of siRNA targeted to MRP1 and BCL2 mRNA for suppression of pump and nonpump cellular resistance in non-small cell lung carcinoma, respectively. Targeting of MSN to cancer cells was achieved by the conjugation of LHRH peptide on the surface of MSN via poly(ethylene glycol) spacer. The delivered anticancer drugs and siRNA preserved their specific activity leading to the cell death induction and inhibition of targeted mRNA. Suppression of cellular resistance by siRNA effectively delivered inside cancer cells and substantially enhanced the cytotoxicity of anticancer drugs. Local delivery of MSN by inhalation led to the preferential accumulation of nanoparticles in the mouse lungs, prevented the escape of MSN into the systemic circulation, and limited their accumulation in other organs. The experimental data confirm that the developed DDS satisfies the major prerequisites for effective treatment of non-small cell lung carcinoma. Therefore, the proposed cancer-targeted MSN-based system for complex delivery of drugs and siRNA has high potential in the effective treatment of lung cancer.

Modular Construction of Fluorescent Lipophosphoramidates by Click Chemistry

AbstractThe synthesis of fluorescent lipophosphoramidates is reported. Their modular construction allows several variations of the molecular structure including the separation of the fluorescent probe from the lipophosphoramidate moiety by a short methylene or by a tetraethylene glycol spacer. The last step of the convergent synthesis is a Huisgen click reaction, which assembles the fluorescent probe with the lipophosphoramidate part. Five fluorescent probes have been considered in this study including coumarin, NBD, fluorescein, naphthalimide and cyanine.