Delivery of Peptide Anti Cancer Drugs

Ras mutations exist in almost 20-30% of human tumors, and the ras oncogenes possess single-point mutations in the sequence coding for the active site of RAS protein at codons 12, 13b, and 61.202 These point mutations are good targets for the antisense oligonucleotides and can suppress the translation and targeted mutant mRNA. The mutated ras genes are involved in the cell proliferation and tumorigenicity. Antisense oligonucleotides (ODN) are molecules that are able to inhibit onco-gene expression, being therefore potentially active for the treatment of viral infections or cancer.

However, because of their poor stability in biological media and their weak intracellular penetration, colloidal drug carriers such as nanoparticles were employed for the delivery of oligonucleotides.203 Association with nanoparticles protects the ODN against degradation and enables them to penetrate more easily into different types of cells. Thus, they were shown to improve the efficiency in inhibiting the proliferation of cells expressing the point-mutated Ha-ras gene. In vivo, the PACA nanoparticles were able to efficiently distribute the ODNs to the liver. As the ODNs have no affinity toward the PACA structure, the ion pair technique was adopted, using cationic surfactants such as cetyltrimethyl ammonium bromide or diethylaminoethyl dextran adsorbed onto the particle surface.204,205 Spontaneous interpolymer complexation between cationic polyelectrolytes and DNA is known and is largely the result of cooperative electrostatic forces.206

ODNs adsorbed onto the PIHCA nanoparticles inhibited the Ha-Ras dependant tumor growth in mice. The ODN-nanoparticle formulation was tested in two cell lines, HBL100rasl and HBL100neo.202 HBL100rasl is a clone obtained from the human mammary cell line. It expresses normal Ha-ras and Ha-ras carrying the G/U point mutation in codon 12 coding for Val instead of Gly at position 12. HBL100neo is a clone transformed only with the pSV2 vector, and it expresses only normal Ha-ras. Three 12-mer oligonucleotides—(1) an antisense oligonucleotide (AS-Val, 50-CACCGACGGCGC-3') directed against and centered at the point mutation in codon 12 of the Ha-ras mRNA, (2) an antisense oligonucleotide (AS-Gly, 50-CACCGCCGGCGC-30) targeted to the equivalent sequence of the normal Ha-ras mRNA, and (3) the 5'/3' inverted sequence of AS-Val that contains the same bases as the antisense sequence but in reverse orientation (INV-Val, 5'-CGCCGGAGCCAC-30). The inhibitory effect of AS-Val adsorbed on the nanoparticles was found to be at a concentration 100-times lower than that for the free AS-Val when tested on HBL100rasl. Cellular uptake studies using 32P-labeling of free ODN and ODN adsorbed nanopar-ticle formulation revealed that the ODNs adsorbed onto the nanoparticles, incorporated at a lower rate than the free ODN but remained intact in the cells even after 72 h post incubation; the free ODN, in contrast, degraded after 3 h. Upon administration into the nude mice treated with HBL100rasl cells, the AS-Val nanoparticle formulation showed more potential tumor growth inhibition compared to the INV-Val nanoparticle formulation, indicating the sequence-dependant effect of ODN on the tumor-inhibition properties.

The cationic surfactants used for the preparation of ODN-PACA nanoparticles may cause toxicity. In addition, the esterase-induced erosion of PACAs causes rapid desorption of ODNs in serum.207 To overcome these drawbacks, the PACA nanocapsules with aqueous core have been

208,209 209

designed. , These nanocapsules enhanced the protection and serum stability of the ODNs. Intratumoral injection of PACA nanoparticles containing Phosphorothioate oligonucleotides directed against EWS Fli-1 chimeric RNA led to a significant tumor growth inhibition compared to the free ODN in mice-bearing Ewing sarcoma.

Peptides like Leu-enkephalin dalargin and the Met-enkephalin kyotorphin normally do not cross the BBB when given systemically. Transport of these neuropeptides across the BBB was achieved by adsorption onto the surface of PBCA nanoparticles and successive coating of the nanoparticles with polysorbate (Tween) 80.210

Birrenbach and Speiser5 described the polymerization process for the preparation of hydro-philic micelles containing solubilized drug molecules including labile proteins in a colloidal aqueous system of dissolved monomers. The hydrocarbon medium constituted the outer phase. After secondary solubilization with the aid of selected surfactants, the polymerization of micelles under different conditions was performed. The entrapped tagged material (human 125I-immuno-globulin G) showed a stable fixation in the nanoparticles during long-term in vitro liberation trials. Nanoparticles were spherical in shape, 80 nm in diameter, and embedded with antigenic material (tetanus toxoid and human immunoglobulin G) for parenteral use. These preparations showed intact biological activity and high antibody production in animals.

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