Detection of endogenous K-ras mRNA in living cells at a single base resolution by a PNA molecular beacon.

Detection of mRNA alterations is a promising approach for identifying biomarkers as means of differentiating benign from malignant lesions. By choosing the KRAS oncogene as a target gene, two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (TO-PNA-MB, where TO = thiazole orange) were synthesized and compared in vitro and in vivo. Their specificity was examined in wild-type KRAS (HT29) or codon 12 point mutation (Panc-1, SW480) cells. Incubation of both beacons with total RNA extracted from the Panc-1 cell line (fully complementary sequence) showed a fluorescent signal for both beacons. Major differences were observed, however, for single mismatch mRNA transcripts in cell lines HT29 and SW480. PS-DNA-MB weakly discriminated such single mismatches in comparison to TO-PNA-MB, which was profoundly more sensitive. Cell transfection of TO-PNA-MB with the aid of PEI resulted in fluorescence in cells expressing the fully complementary RNA transcript (Panc-1) but undetectable fluorescence in cells expressing the K-ras mRNA that has a single mismatch to the designed TO-PNA-MB (HT29). A weaker fluorescent signal was also detected in SW480 cells; however, these cells express approximately one-fifth of the target mRNA of the designed TO-PNA-MB. In contrast, PS-DNA-MB showed no fluorescence in all cell lines tested post PEI transfection. Based on the fast hybridization kinetics and on the single mismatch discrimination found for TO-PNA-MB we believe that such molecular beacons are promising for in vivo real-time imaging of endogenous mRNA with single nucleotide polymorphism (SNP) resolution.

Detecting a secreted gastric cancer biomarker molecule by targeted nanoparticles for real-time diagnostics.

PURPOSE: A real time detection of gastric cancer-associated biomarker molecules in the lumen of the stomach could assist in early detection of this multi-step malignancy. METHODS: Employing α1-antitrypsin precursor (A1AT) as a secreted biomarker model, a platform with immunoassay capabilities, comprising sensing and detecting compartments was developed. It was made of a microarray-type functionalized glass, containing a high density of amine groups. Trypsin, the capturing moiety, was immobilized to the glass surface with the aid of a PEG-based spacer mixture, identified as being crucial for both capturing and detecting properties. The detecting compartment contained near infrared fluorescently labeled nanoparticles conjugated to A1AT-specific antibodies, aimed at generating an optical signal, detectable by a conventional endoscope or a video capsule. RESULTS: The specific recognition reaction between the captured A1AT and the immuno-nanoparticles generated a profound fluorescence with a signal to noise ratio (SNR) of 12-32, in a biomarker-concentration dependent manner. Moreover, the optical recognition signal was intense enough to be detected by a video capsule simulator (with optical detection capabilities of a video capsule) with a SNR of 6-20. CONCLUSIONS: This platform could serve as a real time diagnostic kit for early detection of a secreted biomarker of gastric cancer.

Synchronizing the release rates of topotecan and paclitaxel from a self-eroding crosslinked chitosan - PLGA platform.

This study is a continuation of a previous study in which two model drugs, sodium salicylate (highly water-soluble) and indomethacin (low water-soluble) were loaded into an erodible hydrogel, made of ionically crosslinked chitosan (x-Ct). The erosion rate of the x-Ct matrix was controlled by its immersion in calcium chloride solutions (de-crosslinker) of different concentrations, leading to synchronization of the release rates of the two drugs over 2 h. In the present study, a modified platform was developed in order to (a) synchronize the release rates of the two cytotoxic drugs, topotecan (TT, highly water soluble) and paclitaxel (PTX, poorly water soluble); (b) prolong the erosion duration and the derived concomitant release of the two drugs to several days. TT was loaded into a PLGA sphere, which was co-loaded with calcium chloride (CaCl2). The sphere was then placed in an aqueous solution of chitosan (Ct) in which PTX was dispersed. A PLGA core-containing hydrogel was then produced by ionically crosslinking the Ct. The formulation screening section of the study includes a statistically designed Fractional Factorial experiment. It was comprised of the following five experimental factors: (a) the type of Ct and (b) its relative amount in the formulation, (c) the type of ionic crosslinker (citric acid or oxalic acid), (d) the concentration of the ionic crosslinker and (e) the co-loaded amounts of CaCl2 (the constitutional de-crosslinking agent). The difference factor, f1, and the similarity factor, f2, of the TT and PTX release profiles into water, were used as the experimental responses. The computerized prediction models were employed to assess the collective effects of the pre-determined experimental factors on the difference factor, f1, and the similarity factor, f2 (the response factors), by employing a fractional factorial design and multifactorial analysis, without the need to account for the exact mechanisms of the release processes involved. The final composite platform was capable of releasing TT and PTX, at similar (concomitant) rates, over a period of 7 days, a finding which suggests that the novel polymeric platform may serve as a multi-drug implant. An attractive medical application for such a device would be post-operative local treatment that could benefit from localized combination chemotherapy after the removal of malignant tissues, in the surgical treatment of breast cancer, ovarian cancer, glioma and peritoneal carcinomatosis.

Controlling the release rate of topotecan from PLGA spheres and increasing its cytotoxicity towards glioblastoma cells by co-loading with calcium chloride.

It has been suggested that local administration of topotecan (TT) could increase its efficacy in the treatment of glioblastoma. In this context, a PLGA implant model in the form of spheres with a porous core and stiff surface, loaded with TT and CaCl2 was developed. An array of formulations differing from each other by the type of PLGA used, the integrity of the surface, the concentrations of TT and CaCl2 added during the preparation, and the volume of water in the PLGA mix, was prepared, screened and explored by computerized multifactorial analysis. This analysis enabled the simultaneous identification of the most influential experimental factors on the experimental responses, which were pre-determined as the efficiency of TT loading and the TT % cumulative release at 14 days. The multifactorial analysis also revealed how the interactions among the experimental factors affect the performance of the various formulations. Thus, TT concentration and its factorial interaction with the concentration of CaCl2 added during the spheres' preparation were identified as most prominent on the loading efficiency, while the surface integrity (intact or punctured) and CaCl2 amount in the spheres were identified as most prominent on the TT % cumulative release from the spheres. TT was found to be cytotoxic towards glioblastoma U87 MG cells, an activity which was enhanced, synergistically, in the presence of CaCl2 (the relative viability was reduced from 36 to 28% with combination indices of 1.0, 0.37, 0.13 and 0.06 for EC50, EC75, EC90 and EC95, respectively). Interestingly, dividing the TT dose into 3 equal portions, replenished daily to the incubation medium, increased TT cytotoxicity. The relative viability was then reduced from 35 to 7% and in the presence of CaCl2 - from 28 to 1.9%, suggesting that a local, slow input of TT could be effective in the treatment of glioblastoma by an adjacent TT implant. The increased effect of CaCl2 on cytotoxicity was also observed when it was co-loaded into the TT spheres. In that case, the cells' viability was reduced from 72 to 27%. It is suggested that the PLGA spheres could be used for tunable local delivery of TT in post-resection adjuvant therapy of glioblastoma.

Specific detection of gastric alpha-antitrypsin by immobilized trypsin on polyHEMA films.

Early diagnosis of gastric carcinoma is crucial for maximizing medical treatment efficacy. For the purpose of real time diagnosis ("virtual biopsy") of stomach malignancy we developed a polyHEMA platform capable of capturing human alpha1-antitrypsin precursor (A1AT); a model proteinaceous luminal biomarker. Its specific attachment to the polymeric platform was accomplished by immobilized trypsin, which was linked to the surface of the polyHEMA film by a series of PEG-based spacers. Recognition was enabled by adapting an ELISA-like methodology, using rabbit anti-A1AT and HRP-conjugated anti-rabbit IgG as a secondary antibody. Since this A1AT-sensing platform was designed to be detected by endoscopic means such as a video capsule, its physical stability was tested after casting on top of a polycarbonate surface. It was found that, in contrast to classical ELISA analysis performed on polystyrene plates, A1AT detection was possible only when spacer arms were used to immobilize the capturing moiety, trypsin, with a 7-fold increase in the optical signal and a saturation kinetics dependency upon the concentration of the A1AT biomarker.

Aminoalkylcarbamoylphosphonates reduce TNFα release from activated immune cells.

Some carbamoylphosphonates (CPOs) inhibit matrix metalloproteinases (MMPs). Although MMPs are involved in inflammatory processes, the anti-inflammatory activity of CPOs has not been reported. In this context we compared the biological activity of the three aminoCPOs, PYR-CPO, PIP-CPO and cis-ACCP. We were particularly interested in their capability to modulate the secretion of tumor necrosis factor alpha (TNFα). LPS-activated mouse peritoneal macrophages and LPS-activated mouse splenocytes were used to explore this question. It was found that the aminoCPOs were able to reduce TNFα secretion to a level equivalent to the reduction caused by the steroid drug budesonide (BUD). The reduction in TNFα levels was neither accompanied by cytotoxicity, nor did it inhibit cell proliferation. To explicate whether the aminoCPOs affect TNFα processing by TNFα-converting enzyme (TACE), TACE inhibitory properties of the three molecules was tested in vitro. Only PIP-CPO exerted TACE inhibitory activity at therapeutic (non-cytotoxic) concentrations, indicating on its potential to serve as an anti-inflammatory agent by reducing TNFα secretion.

Mo(CO)6-mediated intramolecular Pauson-Khand reaction of substituted diethyl 3-allyloxy-1-propynylphosphonates.

Cyclisation of diethyl 3-allyloxy-1-propynylphosphonates with Mo(CO)(6) under PK conditions to give 3-substituted-5-oxo-3,5,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-ylphosphonate, 2a-h, in 45-88% isolated yields was done. The R groups are always syn with H(b) (where applicable). The stereochemistry was determined via both NMR and crystal X-ray analysis.

Synthesis of 3-hydroxy-1-alkenylboronates via phosphine stabilized borylzirconacyclopropenes.

Zirconacyclopropenylboronates can be stabilized to dimerization by complexation with tributylphosphine; the phosphine stabilized zirconacycle boronates react with aliphatic and aromatic ketones and aldehydes at C2 of the triple bond to give the previously unknown 3-hydroxyvinylboronates in 61-80% isolated yields.

Synchronizing the release rates of salicylate and indomethacin from degradable chitosan hydrogel and its optimization by definitive screening design.

Three types of ionically crosslinked (with citric acid) chitosan discs were loaded with the highly water- soluble drug, sodium salicylate (SS) and the poorly water-soluble drug, indomethacin (Ind). In separate experiments the hydrated discs were immersed in a de-crosslinking solution comprising of different concentrations of calcium chloride, which induced a controlled erosion of the discs, a process which was optimized to synchronize the release rates of the two drugs over a predetermined period of time. The optimization was accomplished by manipulating six factors: chitosan MW, its amount in the formulation, the concentration of the crosslinker agent, the concentration of the de-crosslinking agent in the dissolution medium, its pH and its temperature. A computerized multifactorial definitive screening design analysis assisted in minimizing the number of experiments. The quotient of the SS to Ind release rates, the difference factor f1, the similarity factor f2 and the combination of f1 and f2 were determined as the experimental responses. The computerized prediction profilers that were used to simulate the contribution of the experimental factors and their effect on the experimental responses led to a successful erodible formulation with a concomitant release of the two drugs over 150 min.

Biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat.

The aim of the present study was to evaluate the toxicity of biodegradable hydrogels in the rat with a future aim of utilizing this hydrogel as a vehicle for brachytherapy delivery in cancer patients. Two types of chitosan hydrogels: fast degrading and slow degrading; were prepared and surgically implanted in rats. The adjacent tissue response to the gels after subcutaneous and intraperitoneal implantation was examined histologically and found to be identical to typical foreign body response and was milder than the response to absorbable surgical sutures (Vicril). Neither tissue damage nor gel fragments could be detected in distant organs (brain, heart, lungs, liver, spleen, kidney, and sternal bone marrow) after implantation of the hydrogels. The degradation mechanism of the gels was studied in vivo, and it was deduced that an oxidative process degraded the chitosan. Loading the hydrogels with a radioisotope (131I-norcholesterol) caused a severe tissue response and necrosis in adjacent tissues only at a distance of several microns. It is concluded that crosslinked chitosan implants could serve as alternative, biocompatible, and safe biodegradable devices for radioisotope delivery in brachytherapy for cancer.

Increasing the affinity of cationized polyacrylamide-paclitaxel nanoparticles towards colon cancer cells by a surface recognition peptide.

Nanoparticles of cationized polyacrylamide (CPAA NP), decorated with the recognition peptide VRPMPLQ (to produce CPAA-Pep NP), were prepared, characterized and tested biologically. They were designed to target dysplasia regions in the colon, characterized by overexpressed sialic acid. This targetability was augmented by the addition of VRPMPLQ. Their mean hydrodynamic size was 137nm with narrow size distribution and positive zeta potential. When incubated with three types of colon cancer cells, a 10-fold increase in the cell's uptake was found for the CPAA-Pep NP compared with the CPAA NP. The use of a scrambled sequence of the VRPMPLQ peptide and competition studies, employing excess of the free peptide verified the specific nature of the NP cellular uptake. Nanoparticles loaded with paclitaxel with and without VRPMPLQ indicated an improved pro-apoptotic activity of the CPAA-Pep NP. It is speculated that both positive charge and the presence of VRPMPLQ could serve as an improved strategy to deliver nanoparticles loaded with cytotoxic drugs for the treatment of colon cancer.

The effect of linker type and recognition peptide conjugation chemistry on tissue affinity and cytotoxicity of charged polyacrylamide.

To increase colonoscopy competence in ambiguous situations (e.g. the existence of flat polyps), an explicit in situ (at real time) diagnosis at the molecular level is required. We have previously shown that the affinity of fluorescent cationic polyacrylamide (Flu-CPAA) to malignant regions in the colon mucosa can be improved by conjugating the recognition peptide EPPT1 to the polymer backbone (to form Flu-CPAA-Pep). Using another recognition peptide, namely VRPMPLQ, we elucidated in the present study the effect of linker type and conjugating methods on Flu-CPAA-VRPMPLQ cytotoxicity and on its affinity to cell lines as well as human colorectal cancer (CRC) biopsies. In order to derive the relationship between the response variable and the experimental factors in a minimal set of experiments, a computerized statistical design of experiment (DoE) strategy was implemented. Data were collected in a six-factor factorial design to study the effect of experimental factors (independent variables) on the ability of the Flu-CPAA polymers to bind specifically to the colon cancer cell lines or the human biopsies (the response). It was found that the presence of VRPMPLQ on the Flu-CPAA improved the polymer's affinity to the human CRC biopsies and to the colon cancer cell lines representing stage B in the Duke severity staging system. The cytotoxicity of Flu-CPAA with high charge density was reduced after conjugated with VRPMPLQ. The replacement of Ahx linker by PEG linker of similar length did not affect the affinity to the human biopsies, nor did it affect cytotoxicity. However, elongating the PEG linker reduced the in vitro affinity to the colon cancer cell lines and to human CRC biopsies. Changing the conjugation method from condensation (amide bond formation) to the click conjugation method did not affect the affinity properties of the polymers. It did reduce, however, the polymer cytotoxicity. We suggest that Flu-CPAA-Pep, with the VRPMPLQ peptide as a recognition moiety, could serve for early diagnosis and screening of CRC patients during endoscopic procedures.

Crosslinked chitosan implants as potential degradable devices for brachytherapy: in vitro and in vivo analysis.

Compared with conventional external beam radiation, brachytherapy offers a superior therapeutic regimen. However, some major constraints are associated with its implementation, including the need of complicated procedures for device placement and removal. The purpose of this study was to examine whether crosslinked chitosan (Ct) implants could serve as potential biodegradable devices for brachytherapy. Ct was reacted with increasing amounts of glutaraldehyde to obtain hydrogels with different crosslinking densities, which were characterized chemically, thermally and mechanically. The effect of the dialysis medium conditions (ionic strength, osmolarity and pH) on the gel hydration and in vivo degradation was assessed. Two types of implants, slow and fast degrading gel (SDG and FDG, respectively), were prepared and implanted with or without Sudan Black (SB) in the rat. While SDG withstood for over a month, the FDG degraded within two weeks after implantation. The release kinetics of SB from the hydrogels verified their in vivo degradation properties. The incorporation of the radioactive compound (131)I-norcholesterol ((131)I-NC) into the SDG altered the degradation kinetics of the gel as reflected by the release kinetics of the radioactive marker. Eighty percent of (131)I-NC was released within a month after implantation, after which time, radioactivity was detected in the regional lymph nodes. Histological examination of the tissues surrounding the implants demonstrated negligible tissue response to the implants, when compared to biodegradable surgical sutures. It is concluded that hydrogels made of crosslinked Ct are potential novel, safe, degradable devices for brachytherapy.

Local treatment of experimental colitis in the rat by negatively charged liposomes of catalase, TMN and SOD.

Superoxide dismutase (SOD), 4-amino tempol (tempamine, denoted as TMN) and catalase were encapsulated into negatively charged liposomes. The activity of the antioxidants against dinitrobenzenesulfonic acid (DNBS) induced colitis was tested in the rat and compared to the anti-inflammatory activity of the native enzymes and free TMN. Inflammation severity was assessed by monitoring tissue myeloperoxidase (MPO) activity, thiobarbituric acid reactive species (TBARS) amounts and by comparing the weights of the dissected colons. In all cases, the liposomal preparations of the antioxidants were more effective than the free molecules in the treatment of the experimental colitis, probably due to the attachment of the negatively charged liposomes, and consequently a longer residence time and better uptake of the antioxidants to the inflamed mucosa. This study suggests that low and high molecular weight antioxidants delivered via anionic liposomes can serve as a novel targeted therapy to treat chronic inflammation of the colonic epithelium, such as ulcerative colitis.

Luminal delivery and dosing considerations of local celecoxib administration to colorectal cancer.

The purpose of this study was to develop a biodegradable drug platform composed of chitosan and guar gum and to explore the possibility of using it for local adjuvant or neoadjuvant therapy of colorectal cancer. Celecoxib (Cx), a chemopreventative drug for familial adenomatous polyposis (FAP) and under trial for reducing post surgical colorectal malignancies, was selected as a model drug for this topical system because of the contraindications that are associated with its systemic administration. Films made of chitosan (Ct) and guar gum (GG) were prepared, characterized for equilibrium swelling, mucoadhesion, in vitro and in vivo degradation and loaded with Cx. Short term dosing studies in vitro were performed in the HT-29 colon carcinoma cell line that was incubated with Cx using the MTT test to assess IC50. The impact of a single high dose was evaluated and compared with a repeating low-dose regimen. In vivo dosing experiments with Cx were performed in the perfused intestine of the anaesthetized rat. Measuring tissue LDH assessed epithelium injury. Mechanical, mucoadhesion and in vitro degradation of the polysaccharide films were dictated by manipulating the ratios of Ct and GG. The addition of rat cecal contents to the dissolution medium increased the total Cx released from those films containing high amounts of GG. MTT reduction, a measure of cell proliferation, diminished as a function of increasing drug concentration and exposure time in the HT-29 cell line studies. Local high concentrations of Cx were shown to impede the proliferation of cancer cells directly, while chemoprevention has been demonstrated with low Cx doses. Healthy cells were shown to be sensitive to high Cx doses. Maximum therapeutic efficiency in the context of minimal healthy tissue exposure would thus be predicted utilizing a local delivery system such as the proposed adhesive, biodegradable polysaccharide composites.

Targeting normal and neoplastic tissues in the rat jejunum and colon with boronated, cationic acrylamide copolymers.

A series of boronated cationic copolymers, composed of different ratios of acrylamide, N-acryloyl-3-aminophenylboronic acid and N-acryloyl-diaminoethane (the cationic moiety), were prepared with the intention of localizing boron neutron capture therapy (BNCT) in experimentally induced polyps on the luminal side of the gut of the rat. The goals of this study were to: (a) test the effect of cationization of the boronated copolymers on their uptake by polyps and normal adjacent epithelium; (b) compare the whole rat body distribution of aminophenylboronic acid (APB) and polymeric APB after local application; (c) measure the effect of micro-environmental parameters such as pH, the presence of mucin and cations on the interaction between the APB-copolymers and the epithelium of the rat intestines. Direct analysis of tissue boron levels showed that polymeric APB-uptake was higher in the colonic polyps than in the surrounding normal tissues. Free APB, however, was found in similar quantities in both. When tested in the normal jejunum and colon of the rat, polymeric APB uptake was directly proportional to the molar content of the cationic monomer in the copolymers. The presence of magnesium ions, free boron cationic monomer and mucin interfered with this uptake in a concentration-dependent manner. The uptake was pH-independent at pH 5, 7 and 10. APB accumulation in the colon polyps was inversely proportional to the cationic monomer content in the copolymers, suggesting an increased amount of mucus around the polyps, which probably impeded the electrostatic attachment of the polymer to the malignant tissue. The use polymeric APB for targeting BNCT in perioperative treatment of colorectal carcinoma is suggested, especially in the cases of microscopic residual disease after curative resection.

Colonic drug delivery.

Fifteen years of research in the area of colon-specific drug delivery has left us with a slim choice of viable techniques, not because of the lack of proofs of concept but because of the ambiguity regarding the therapeutic necessity of targeting the colon with drugs. Critical analysis of existing technologies as well as medically based novel ideas could lead to interesting prospects.:

The anti-inflammatory activity of a novel fused-cyclopentenone phosphonate and its potential in the local treatment of experimental colitis.

A novel fused-cyclopentenone phosphonate compound, namely, diethyl 3-nonyl-5-oxo-3,5,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-ylphosphonate (P-5), was prepared and tested in vitro (LPS-activated macrophages) for its cytotoxicity and anti-inflammatory activity and in vivo (DNBS induced rat model) for its potential to ameliorate induced colitis. Specifically, the competence of P-5 to reduce TNFα, IL-6, INFγ, MCP-1, IL-1α, MIP-1α, and RANTES in LPS-activated macrophages was measured. Experimental colitis was quantified in the rat model, macroscopically and by measuring the activity of tissue MPO and iNOS and levels of TNFα and IL-1ß. It was found that P-5 decreased the levels of TNFα and the tested proinflammatory cytokines and chemokines in LPS-activated macrophages. In the colitis-induced rat model, P-5 was effective locally in reducing mucosal inflammation. This activity was equal to the activity of local treatment with 5-aminosalicylic acid. It is speculated that P-5 may be used for the local treatment of IBD (e.g., with the aid of colon-specific drug platforms). Its mode of action involves inhibition of the phosphorylation of MAPK ERK but not of p38 and had no effect on IκBα.

Multi-modal detection of colon malignancy by NIR-tagged recognition polymers and ultrasound contrast agents.

To increase colonoscopy capability to discriminate benign from malignant polyps, we suggest combining two imaging approaches based on targeted polymeric platforms. Water-soluble cationized polyacrylamide (CPAA) was tagged with the near infrared (NIR) dye IR-783-S-Ph-COOH to form Flu-CPAA. The recognition peptide VRPMPLQ (reported to bind specifically to CRC tissues) was then conjugated with the Flu-CPAA to form Flu-CPAA-Pep which was then incorporated into echogenic microbubbles (MBs) made of polylactic acid (PLA) that are highly responsive to ultrasound. The ultimate design includes intravenous administration combined with local ultrasound and intra-colon inspection at the NIR range. In this proof of principle study PLA MBs were prepared by the double emulsion technique and loaded with several types of Flu-CPAA-Pep polymers. After insonation the submicron PLA fragments (SPF)-containing Flu-CPAA-Pep were examined in vitro for their ability to attach to colon cancer cells and in vivo (DMH induced rat model) for their ability to attach to colon malignant tissues and compared to the specific attachment of the free Flu-CPAA-Pep. The generation of SPF-containing Flu-CPAA-Pep resulted in a tissue attachment similar to that of the free, unloaded Flu-CPAA-Pep. The addition of VRPMPLQ to the polymeric backbone of the Flu-CPAA reduced cytotoxicity and improved the specific binding.