Turning stealth liposomes into cationic liposomes for anticancer drug delivery

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0

Imidazole-Based pH-Sensitive Convertible Liposomes for Anticancer Drug Delivery

In efforts to enhance the activity of liposomal drugs against solid tumors, three novel lipids that carry imidazole-based headgroups of incremental basicity were prepared and incorporated into the membrane of PEGylated liposomes containing doxorubicin (DOX) to render pH-sensitive convertible liposomes (ICL). The imidazole lipids were designed to protonate and cluster with negatively charged phosphatidylethanolamine-polyethylene glycol when pH drops from 7.4 to 6.0, thereby triggering ICL in acidic tumor interstitium. Upon the drop of pH, ICL gained more positive surface charges, displayed lipid phase separation in TEM and DSC, and aggregated with cell membrane-mimetic model liposomes. The drop of pH also enhanced DOX release from ICL consisting of one of the imidazole lipids, sn-2-((2,3-dihexadecyloxypropyl)thio)-5-methyl-1H-imidazole. ICL demonstrated superior activities against monolayer cells and several 3D MCS than the analogous PEGylated, pH-insensitive liposomes containing DOX, which serves as a control and clinical benchmark. The presence of cholesterol in ICL enhanced their colloidal stability but diminished their pH-sensitivity. ICL with the most basic imidazole lipid showed the highest activity in monolayer Hela cells; ICL with the imidazole lipid of medium basicity showed the highest anticancer activity in 3D MCS. ICL that balances the needs of tissue penetration, cell-binding, and drug release would yield optimal activity against solid tumors

Turning stealth liposomes into cationic liposomes for anticancer drug delivery

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0

Synthesis and characterization of a hydrazone based pH-sensitive lipid polymer conjugate

A three-step synthesis of a lipid-polymer conjugate is presented. The lipid, 1,2-di-O-hexadecyl-rac-glycerol (DHG) is conjugated to polyethylene glycol 2000 (PEG2000) via a pH-sensitive hydrazone linker. The steps involved in the synthesis of the conjugate include 1) incorporation of an ester group on the DHG moiety 2) hydrozinolysis of the ester group to yield a DHG hydrazide 3) condensation of the DHG hydrazide with m-PEG2000 aldehyde to yield the DHG-Hydrazone-PEG2000 conjugate. This hydrazone-based conjugate shows a pH-dependent hydrolysis and can be incorporated into liposomes to trigger cargo release at low pH environments as in cancer tissues

Key Design Features of Lipid Nanoparticles and Electrostatic Charge-Based Lipid Nanoparticle Targeting

Lipid nanoparticles (LNP) have gained much attention after the approval of mRNA COVID-19 vaccines. The considerable number of currently ongoing clinical studies are testament to this fact. These efforts towards the development of LNPs warrant an insight into the fundamental developmental aspects of such systems. In this review, we discuss the key design aspects that confer efficacy to a LNP delivery system, i.e., potency, biodegradability, and immunogenicity. We also cover the underlying considerations regarding the route of administration and targeting of LNPs to hepatic and non-hepatic targets. Furthermore, since LNP efficacy is also a function of drug/nucleic acid release within endosomes, we take a holistic view of charged-based targeting approaches of LNPs not only in the context of endosomal escape but also in relation to other comparable target cell internalization strategies. Electrostatic charge-based interactions have been used in the past as a potential strategy to enhance the drug release from pH-sensitive liposomes. In this review, we cover such strategies around endosomal escape and cell internalization in low pH tumor micro-environments

DESIGN OF PH-TURNABLE LIPOSOMES FOR ANTICANCER DRUG DELIVERY

Liposomal drug delivery inside the tumor cells has been a challenge due to either recognition of liposomes by reticuloendothelial cells or due to inefficient drug release from liposomes at the tumor site. We have strived to design and characterize imidazole-based pH-tunable liposomes composed of pH-titrable imidazole-based lipids (C-16 side chains), negatively charged DPPE-PEG (C-16 side chains) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, C-18 side chains). The membrane of each liposome remains as one phase at pH 7.4 where all the lipids comprising the formulation are homogenously distributed. As the pH decreases to 6.0, the imidazole-based lipids protonate and associate with negatively charged DPPE-PEG to form a two-phase liposome membrane, one phase rich in imidazole and DPPE-PEG lipids and the other rich in DSPC. The phase separation on the liposome membrane is driven by electrostatic interactions and vander waals interactions among lipids of similar chain length. The phase-separated liposomes show increased binding and aggregation with negatively charged model liposomes mimicking the cell membrane

Synthesis and characterization of a hydrazone based pH-sensitive lipid polymer conjugate

A three-step synthesis of a lipid-polymer conjugate is presented. The lipid, 1,2-di-O-hexadecyl-rac-glycerol (DHG) is conjugated to polyethylene glycol 2000 (PEG2000) via a pH-sensitive hydrazone linker. The steps involved in the synthesis of the conjugate include 1) incorporation of an ester group on the DHG moiety 2) hydrozinolysis of the ester group to yield a DHG hydrazide 3) condensation of the DHG hydrazide with m-PEG2000 aldehyde to yield the DHG-Hydrazone-PEG2000 conjugate. This hydrazone-based conjugate shows a pH-dependent hydrolysis and can be incorporated into liposomes to trigger cargo release at low pH environments as in cancer tissues

DESIGN OF PH-TURNABLE LIPOSOMES FOR ANTICANCER DRUG DELIVERY

Liposomal drug delivery inside the tumor cells has been a challenge due to either recognition of liposomes by reticuloendothelial cells or due to inefficient drug release from liposomes at the tumor site. We have strived to design and characterize imidazole-based pH-tunable liposomes composed of pH-titrable imidazole-based lipids (C-16 side chains), negatively charged DPPE-PEG (C-16 side chains) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, C-18 side chains). The membrane of each liposome remains as one phase at pH 7.4 where all the lipids comprising the formulation are homogenously distributed. As the pH decreases to 6.0, the imidazole-based lipids protonate and associate with negatively charged DPPE-PEG to form a two-phase liposome membrane, one phase rich in imidazole and DPPE-PEG lipids and the other rich in DSPC. The phase separation on the liposome membrane is driven by electrostatic interactions and vander waals interactions among lipids of similar chain length. The phase-separated liposomes show increased binding and aggregation with negatively charged model liposomes mimicking the cell membrane

Quantitative analysis of nicotine content in commercially available brands of cigarettes

Cigarette smoking continues to plague the modern generation with severe health problems, mostly arising from nicotine. With some leading cigarette brands reportedly increasing the nicotine content on an annual basis, the need for a highly selective extraction and quantification method of nicotine from cigarette tobacco appears critically important. We have developed a method for quantitative analysis of nicotine in cigarettes which is simple yet highly selective for nicotine. We found the nicotine content in three leading cigarette brands viz Marlboro Golden Yellow, Sonoma regular and Camel regular to be 1.7, 1.31 and 0.61 mg per cigarette respectively

Quantitative analysis of nicotine content in commercially available brands of cigarettes

Cigarette smoking continues to plague the modern generation with severe health problems, mostly arising from nicotine. With some leading cigarette brands reportedly increasing the nicotine content on an annual basis, the need for a highly selective extraction and quantification method of nicotine from cigarette tobacco appears critically important. We have developed a method for quantitative analysis of nicotine in cigarettes which is simple yet highly selective for nicotine. We found the nicotine content in three leading cigarette brands viz Marlboro Golden Yellow, Sonoma regular and Camel regular to be 1.7, 1.31 and 0.61 mg per cigarette respectively