Periodic Reporting for period 1 - NanoDeliveryEnhance (Novel functionalization of liposomic nano-vehicles for strongly-enhanced drug delivery)
Período documentado: 2023-02-01 hasta 2024-07-31
The problem we are addressing concerns delivery of drugs via nanoparticles, in particular via liposomes which are sub-micron-sized (ca. 100 nm) vesicles whose membranes are lipid bilayers (similar to the membranes of cells). The delivery of pharmaceutical agents encapsulated in such nanocarriers has numerous advantages, and liposomes are well-established nanocarriers that are used for both diagnostics and treatments. To date, almost all FDA-approved liposomal vehicles are functionalized with poly(ethylene glycol), PEG, in order to be effective for in-vivo applications. This current gold-standard method provides both steric stability and, more crucially, enhances blood retention time and consequently therapeutic efficiency. However, such PEGylation has a number of disadvantages, including decreased cellular uptake, polymer accumulation in tissues, and faster blood clearance on repeated intravenous dosing due to activation of immune response.
In this project, our objectives are to improve currently available nanodelivery vehicles by developing a novel functionalization. Specifically, we have the following aims:
1) to show that replacing PEG with a new phosphorylcholine-based conjugate (pMPC), termed pMPCylation (as opposed to PEGylation), delivers comparable colloidal stability and high biocompatibility
2) enhances cellular uptake and
3) decreases immune response in-vivo, resulting in longer blood circulation times.
As our project has demontrated, indeed the innovative pMPCylation of liposomes shows that it is a safer and more effective alternative to PEGylation, potentially enabling improved lipid-based therapeutics for a wide range of diseases.
In this project, our objectives are to improve currently available nanodelivery vehicles by developing a novel functionalization. Specifically, we have the following aims:
1) to show that replacing PEG with a new phosphorylcholine-based conjugate (pMPC), termed pMPCylation (as opposed to PEGylation), delivers comparable colloidal stability and high biocompatibility
2) enhances cellular uptake and
3) decreases immune response in-vivo, resulting in longer blood circulation times.
As our project has demontrated, indeed the innovative pMPCylation of liposomes shows that it is a safer and more effective alternative to PEGylation, potentially enabling improved lipid-based therapeutics for a wide range of diseases.
We established that pMPC functionalization of liposomic nanodelvery vehicles had the following beneficial characteristics:
1.Colloidal stability of pMPC- liposomes in serum-enriched medium:
A zwitterionic polymer-lipid conjugate, poly(2-methacryloyloxyethyl phosphorylcholine)-distearylphospatidylchoilne (pMPC - DSPE), was used to functionalize our liposomes; it results in biocompatibility and colloidal stability comparable to liposomes functionalized with the current gold-standard PEG-DSPE. We prepared liposomes (large unilamellar vesicles, LUVs) composed of HSPC with either 2% or 5% (mol/mol) PEG or pMPC of varying polymer length, which were also doped with positively charged stearylamine (5% mol/mol) to compensate for the negative charge from functionalized DSPE.
Firstly, we explored the interaction of pMPC moieties with biomolecules typically encountered by nanoparticle delivery vehicles under physiological conditions, and how it affects structural changes and aggregation of the functionalized liposomes. Such interaction is crucial for nanoparticle-based drug carriers, as once the materials come into contact with body fluids via intravenous injection into bloodstream, they are confronted with a high amount of proteins which might create a so-called protein corona, contributing to its biological identity and playing a strong role on nanoparticle-cell interaction and cell uptake.. We therefore determined the effect of protein-rich medium on the stability of the functionalized LUVs colloidal stability, and found it entirely satisfactory.
2. Cellular uptake of pMPC-grafted liposomes:
Following evaluation of pMPC effect on the carriers’ colloidal stability as in (1) above, we determined the efficiency of liposomal cell uptake by regulating the polymer length and lipid composition. Vero cells were selected as target cells, being a common model for such studies as well as for viral infection and antiviral treatment. Our results show indeed that pMPCylation positively affects cellular uptake compared to PEG, with a significant dependence on the polymer length. Specifically, we observe that PEGylated liposomes have little dependence on polymer length. In contrast, pMPCylated LUVs display a clear decrease with increasing polymer length, . Importantly, comparison between pMPC and PEG at the same polymer molecular weight clearly shows that pMPCylation results in much higher internalization by the cells. While some differences in cellular uptake could be partially ascribed to the improved carrier stability with increasing polymer length, PEGylated and pMPCylated liposomes, while possessing similar stability, show significantly different uptake behavior. Overall, these results suggest that the enhanced uptake of pMPCylated carriers reflect a molecular interaction between MPC moieties and the cell membrane, rather than a feature arising from good colloidal properties or carrier stability. In particular, the differences in between PEG and pMPC indicate that the zwitterionic nature of MPC moieties might be the primary cause of such enhanced interaction.
3. Prolonged in-vivo circulation of pMPC- functionalized liposomes:
To evaluate the in-vivo properties of pMPC, liposomes were fluorescently labelled and injected into mice via tail vein; at specific time points, blood was collected and the fluorescence signal in the bloodstream was measured.
The resulting blood circulation profiles and comparison between pMPC and PEG functionalization shows that’s pMPCylation above 5 kDa results in at least 1.5 larger circulation half-time compared to PEG-LUVs. We additionally verified that injection of both PEG and pMPC LUVs would be biocompatible by monitoring the health of the mice. Our results demonstrate that both PEG- and pMPC-liposomes are accumulated primarily in the spleen at all polymer lengths, in agreement with previous reports of biodistribution of PEGylated carriers. The higher cellular uptake of pMPCylated compared to PEGylated liposomes has been proposed to arise from high affinity of phosphorylcholine groups for scavenger receptors that are expressed in cells and induces internalization via endocytosis. Positively charged lipids (SA) added to the LUVs membrane counteract electrostatic repulsion between the negatively charged LUVs and cellular membranes, while pMPC polymer length regulates the balance between PC moieties interaction, and steric repulsion that acts to suppress cellular endocytosis. An optimal balance between the two factors, i.e the intermediate 5 kDa-pMPC, is long enough to provide colloidal stability but at the same provides the enhanced interaction.
4. Immune response against pMPC-liposomes in mice:
Multiple injections of PEGylated liposomes lead to rapid clearance of second and subsequent doses of liposomes (the ABC effect), presumably through activation of the complement system followed by opsonization of the liposomes and consequent secretion of IgM antibodies and activation of the mononuclear system. To examine this hypothesis more directly, we investigated the correlation to IgM antibody secretion in response to a single injection of either PEGylated or pMPCylated liposomes. Our results provide insight into the overall efficiency of these novel functionalized nanodelivery vehicles..
1.Colloidal stability of pMPC- liposomes in serum-enriched medium:
A zwitterionic polymer-lipid conjugate, poly(2-methacryloyloxyethyl phosphorylcholine)-distearylphospatidylchoilne (pMPC - DSPE), was used to functionalize our liposomes; it results in biocompatibility and colloidal stability comparable to liposomes functionalized with the current gold-standard PEG-DSPE. We prepared liposomes (large unilamellar vesicles, LUVs) composed of HSPC with either 2% or 5% (mol/mol) PEG or pMPC of varying polymer length, which were also doped with positively charged stearylamine (5% mol/mol) to compensate for the negative charge from functionalized DSPE.
Firstly, we explored the interaction of pMPC moieties with biomolecules typically encountered by nanoparticle delivery vehicles under physiological conditions, and how it affects structural changes and aggregation of the functionalized liposomes. Such interaction is crucial for nanoparticle-based drug carriers, as once the materials come into contact with body fluids via intravenous injection into bloodstream, they are confronted with a high amount of proteins which might create a so-called protein corona, contributing to its biological identity and playing a strong role on nanoparticle-cell interaction and cell uptake.. We therefore determined the effect of protein-rich medium on the stability of the functionalized LUVs colloidal stability, and found it entirely satisfactory.
2. Cellular uptake of pMPC-grafted liposomes:
Following evaluation of pMPC effect on the carriers’ colloidal stability as in (1) above, we determined the efficiency of liposomal cell uptake by regulating the polymer length and lipid composition. Vero cells were selected as target cells, being a common model for such studies as well as for viral infection and antiviral treatment. Our results show indeed that pMPCylation positively affects cellular uptake compared to PEG, with a significant dependence on the polymer length. Specifically, we observe that PEGylated liposomes have little dependence on polymer length. In contrast, pMPCylated LUVs display a clear decrease with increasing polymer length, . Importantly, comparison between pMPC and PEG at the same polymer molecular weight clearly shows that pMPCylation results in much higher internalization by the cells. While some differences in cellular uptake could be partially ascribed to the improved carrier stability with increasing polymer length, PEGylated and pMPCylated liposomes, while possessing similar stability, show significantly different uptake behavior. Overall, these results suggest that the enhanced uptake of pMPCylated carriers reflect a molecular interaction between MPC moieties and the cell membrane, rather than a feature arising from good colloidal properties or carrier stability. In particular, the differences in between PEG and pMPC indicate that the zwitterionic nature of MPC moieties might be the primary cause of such enhanced interaction.
3. Prolonged in-vivo circulation of pMPC- functionalized liposomes:
To evaluate the in-vivo properties of pMPC, liposomes were fluorescently labelled and injected into mice via tail vein; at specific time points, blood was collected and the fluorescence signal in the bloodstream was measured.
The resulting blood circulation profiles and comparison between pMPC and PEG functionalization shows that’s pMPCylation above 5 kDa results in at least 1.5 larger circulation half-time compared to PEG-LUVs. We additionally verified that injection of both PEG and pMPC LUVs would be biocompatible by monitoring the health of the mice. Our results demonstrate that both PEG- and pMPC-liposomes are accumulated primarily in the spleen at all polymer lengths, in agreement with previous reports of biodistribution of PEGylated carriers. The higher cellular uptake of pMPCylated compared to PEGylated liposomes has been proposed to arise from high affinity of phosphorylcholine groups for scavenger receptors that are expressed in cells and induces internalization via endocytosis. Positively charged lipids (SA) added to the LUVs membrane counteract electrostatic repulsion between the negatively charged LUVs and cellular membranes, while pMPC polymer length regulates the balance between PC moieties interaction, and steric repulsion that acts to suppress cellular endocytosis. An optimal balance between the two factors, i.e the intermediate 5 kDa-pMPC, is long enough to provide colloidal stability but at the same provides the enhanced interaction.
4. Immune response against pMPC-liposomes in mice:
Multiple injections of PEGylated liposomes lead to rapid clearance of second and subsequent doses of liposomes (the ABC effect), presumably through activation of the complement system followed by opsonization of the liposomes and consequent secretion of IgM antibodies and activation of the mononuclear system. To examine this hypothesis more directly, we investigated the correlation to IgM antibody secretion in response to a single injection of either PEGylated or pMPCylated liposomes. Our results provide insight into the overall efficiency of these novel functionalized nanodelivery vehicles..
The potential of our results for improved nanodelivery vehicles is clear, and some of the results contributed to a patent application on the used of our functionalized vesicles as novel drug carriers. For further success, our preliminary studies on a murine model need to be extended, while suitable licensing of our patent and further development and trials by commercial entities will ensure it further development and bringing to market.