Concomitant Chemoradiation Therapy with Gold Nanoparticles and Platinum Drugs Co-Encapsulated in Liposomes

A liposomal formulation of gold nanoparticles (GNPs) and carboplatin, named LipoGold, was produced with the staggered herringbone microfluidic method. The radiosensitizing potential of LipoGold and similar concentrations of non-liposomal GNPs, carboplatin and oxaliplatin was evaluated in vitro with the human colorectal cancer cell line HCT116 in a clonogenic assay. Progression of HCT116 tumor implanted subcutaneously in NU/NU mice was monitored after an irradiation of 10 Gy combined with either LipoGold, GNPs or carboplatin injected directly into the tumor by convection-enhanced delivery. Radiosensitization by GNPs alone or carboplatin alone was observed only at high concentrations of these compounds. Furthermore, low doses of carboplatin alone or a combination of carboplatin and GNPs did not engender radiosensitization. However, the same low doses of carboplatin and GNPs administered simultaneously by encapsulation in liposomal nanocarriers (LipoGold) led to radiosensitization and efficient control of cell proliferation. Our study shows that the radiosensitizing effect of a combination of carboplatin and GNPs is remarkably more efficient when both compounds are simultaneously delivered to the tumor cells using a liposomal carrier

Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development

The development of copper-drug complexes (CDCs) is hindered due to their very poor aqueous solubility. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anticancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)(2)), a highly insoluble complex that has not been possible to develop for indications requiring parenteral administration. We have resolved this issue by synthesizing Cu(DDC)(2) inside liposomes. DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method is successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461. Our method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anticancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients

Vascular normalization in orthotopic glioblastoma following intravenous treatment with lipid-based nanoparticulate formulations of irinotecan (Irinophore C™), doxorubicin (Caelyx®) or vincristine

Background: Chemotherapy for glioblastoma (GBM) patients is compromised in part by poor perfusion in the tumor. The present study evaluates how treatment with liposomal formulation of irinotecan (Irinophore C™), and other liposomal anticancer drugs, influence the tumor vasculature of GBM models grown either orthotopically or subcutaneously. Methods Liposomal vincristine (2 mg/kg), doxorubicin (Caelyx®; 15 mg/kg) and irinotecan (Irinophore C™; 25 mg/kg) were injected intravenously (i.v.; once weekly for 3 weeks) in Rag2M mice bearing U251MG tumors. Tumor blood vessel function was assessed using the marker Hoechst 33342 and by magnetic resonance imaging-measured changes in vascular permeability/flow (Ktrans). Changes in CD31 staining density, basement membrane integrity, pericyte coverage, blood vessel diameter were also assessed. Results The three liposomal drugs inhibited tumor growth significantly compared to untreated control (p < 0.05-0.001). The effects on the tumor vasculature were determined 7 days following the last drug dose. There was a 2-3 fold increase in the delivery of Hoechst 33342 observed in subcutaneous tumors (p < 0.001). In contrast there was a 5-10 fold lower level of Hoechst 33342 delivery in the orthotopic model (p < 0.01), with the greatest effect observed following treatment with Irinophore C. Following treatment with Irinophore C, there was a significant reduction in Ktrans in the orthotopic tumors (p < 0.05). Conclusion The results are consistent with a partial restoration of the blood-brain barrier following treatment. Further, treatment with the selected liposomal drugs gave rise to blood vessels that were morphologically more mature and a vascular network that was more evenly distributed. Taken together the results suggest that treatment can lead to normalization of GBM blood vessel the structure and function. An in vitro assay designed to assess the effects of extended drug exposure on endothelial cells showed that selective cytotoxic activity against proliferating endothelial cells could explain the effects of liposomal formulations on the angiogenic tumor vasculature.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofPharmaceutical Sciences, Faculty ofOther UBCNon UBCReviewedFacult

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats

Abstract Background Effectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood–brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed. Results The cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4′ liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals. Conclusions Selection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations

A simple passive equilibration method for loading carboplatin into pre-formed liposomes incubated with ethanol as a temperature dependent permeability enhancer.

A passive equilibration method which relies on addition of candidate drugs to pre-formed liposomes is described as an alternative method for preparing liposome encapsulated drugs. The method is simple, rapid and applicable to liposomes prepared with high (45mol%) or low (&lt;20mol%) levels of cholesterol. Passive equilibration is performed in 4-steps: (i) formation of liposomes, (ii) addition of the candidate drug to the liposomes in combination with a permeability enhancing agent, (iii) incubation at a temperature that facilitates diffusion of the added compound across the lipid bilayer, and (iv) quenching the enhanced membrane permeability by reduction in temperature and/or removal of the permeabilization enhancer. The method is fully exemplified here using ethanol as the permeabilization enhancer and carboplatin (CBDCA) as the drug candidate. It is demonstrated that ethanol can be added to liposomes prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and Cholesterol (Chol) (55:45mol ratio) in amounts up to 30% (v/v) with no change in liposome size, even when incubated at temperatures&gt;60°C. Super-saturated solutions of CBDCA (40mg/mL) can be prepared at 70°C and these are stable in the presence of ethanol even when the temperature is reduced to &lt;30°C. maximum CBDCA encapsulation is achieved within 1h after the CBDCA solution is added to pre-formed DSPC/Chol liposomes in the presence of 30% (v/v) ethanol at 60°C. When the pre-formed liposomes are mixed with ethanol (30% v/v) at or below 40°C, the encapsulation efficiency is reduced by an order of magnitude. The method was also applied to liposomes prepared from other compositions include a cholesterol free formulations (containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG2000)) and a low Chol (&lt;20mol%) formulations prepared with the distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) DSPG)). The cytotoxic activity of CBDCA was unaffected when prepared in this manner and two of the resultant formulations exhibited good stability in vitro and in vivo. The cytotoxic activity of CBDCA was unaffected when prepared in this manner and the resultant formulations exhibited good stability in vitro and in vivo. Pharmacokinetics studies in CD-1 mice indicated that the resulting formulations increased the circulation half life of the associated CBDCA significantly (AUC0-24h of CBDCA=0.016μg·hr/mL; AUC0-24h of the DSPC/Chol CBDCA formulation=1014.0μg·hr/mL and AUC0-24h of the DSPC/DSPG/Chol CBDCA formulation=583.96μg·hr/mL). Preliminary efficacy studies in Rag-2M mice with established subcutaneous H1975 and U-251 tumors suggest that the therapeutic activity of CBDCA is improved when administered in liposomal formulations. The encapsulation method described here has not been disclosed previously and will have broad applications to drugs that would normally be encapsulated during liposome manufacturing

The anticancer activity of diethyldithiocarbmamate (DDC) is dependent on copper.

<p><b>(A)</b> Disulfiram is metabolized to diethyldithiocarbamate (DDC) and DDC complexes with Copper (Cu) (II). <b>(B)</b> Cytotoxicity curves for DSF (●) and DSF + CuSO₄ (■) were obtained with the IN CELL Analyzer using U87 glioblastoma cells where cell viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. <b>(C)</b> Cytotoxicity curves for DDC (●) and DDC + CuSO₄ (■); where cytotoxicity was measured as described above. <b>(D)</b> DDC and Cu(DDC)₂ IC₅₀ for U251, MDA-231-BR, and A549 cancer cell lines as well as HBEcP a normal cell line; averages (±SEM) are reported from three separate experiments each done in triplicate. <b>(E)</b> Photograph of DDC, CuSO₄ and Cu(DDC)₂ solutions in water.</p

Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO₄.

<p><b>(A)</b> Photograph of solutions consisting of DDC (5mg/mL) and added to CuSO₄-containing DSPC/Chol (55:45) liposomes (20 mM liposomal lipid) over a 1 hour at 25°C. <b>(B)</b> Formation of Cu(DDC)₂ inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hour at 4(●), 25(■) and 40(▲)°C following addition of DDC at a final DDC concentration of (5 mM); Cu(DDC)₂ was measured using a UV-Vis spectrophotometer and lipid was measured using scintillation counting. <b>(C)</b> Cu(DDC)₂ formation inside DSPC/Chol (55:45) liposomes over time where the external pH was 7.4 (▲) and 3.5 (▼). <b>(D)</b> Measured Cu(DDC)₂ as a function of increasing DDC added, represented as the theoretical Cu(DDC)₂ to total liposomal lipid ratio; where the lipid concentration was fixed at 20 mM and final DDC concentration was varied. <b>(E)</b> Cryo-electron microscopy photomicrograph of CuSO₄- containing DSPC/Chol (55:45) liposomes and the same liposomes after formation of encapsulated Cu(DDC)₂. <b>(F)</b> Size of the CuSO₄- containing liposomes and liposomes with encapsulated Cu(DDC)₂ as determined by quasi-electric light scattering and cryo-electron microscopy; data points are given as mean ± SD.</p