Orthogonal Click Conjugation to the Liposomal Surface Reveals the Stability of the Lipid Anchorage as Crucial for Targeting

Abstract

Synthetic access to multiple surface decorations are a bottleneck in the development of liposomes for receptor mediated targeting. This opens a complex multiparameter space, exploration of which is severely limited in terms of sample numbers and turnaround times. Here, we unlock this technological barrier by a combination of a milligram-scale liposome formulation using dual centrifugation and orthogonal click chemistry on the liposomal surface. Application of these techniques to conceptually new amphiphilic compounds, which feature norbornene and alkyne groups at the apex of sterically stabilizing, hyperbranched polyglycerol moieties, revealed a particular influence of the membrane anchor of functional amphiphiles. Folic acid residues clicked to cholesterol-based amphiphiles were inefficient in folate-mediated cell targeting, while dialkyl-anchored amphiphiles remained stable in the liposomal membrane and imparted efficient targeting properties. These findings are of specific importance considering the popularity of cholesterol as a lipophilic anchor.

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell‐Surface Anchoring of Multivalent Antibody‐Recruiting Polymers

“…[21] Cholesterol-based conjugates have been reported to undergo fast exchange from the cell membrane,w hich might account for the less efficient antibody-recruiting capacity of cholesterol-based ARPs. [22] Using the dialkyl-polyDNP conjugate, we subsequently tested its persistence on the cell surface over prolonged periods of time by treating cells with polymers followed by pulsing with anti-DNP at different time points prior to flow cytometry and confocal microscopy analysis.I n these experiments,c ells were carefully washed to remove unbound polymer to avoid continuous replenishing of lipidpolymer on the cell surface from lipid-polymer in solution. These experiments ( Figure 2E)d emonstrate that for at least 4days post dialkyl-polyDNP treatment, anti-DNP can bind to the cell surface.W hile antibody-dependent effector mechanisms occur fairly rapidly (see below for ADCP data), we hypothesize that prolonged cell surface display in vivo of ARPs allows for ahigher chance of Fc-recognition to occur and innate effector killing to take place.M oreover, our data show that despite the fact that ARPs become endocytosed to ac ertain extent (as evidenced by the dotted red fluorescent intracellular pattern in the confocal images in Interestingly,e arlier reports suggest the cell surface persistence of dialkyl-polymer conjugates to be no more than 24 h, [19,20] whereas at present we demonstrate antibody recruitment for at least 4days on dialkyl-polyDNP-treated cells.H owever,i nt hese previous works,t ypically phospholipid-based conjugates were used whereas in our present work we use neutral charged amide-linked lipids.T hese findings highlight the complex influence of lipid-polymer amphiphile structure and membrane anchoring and internalization kinetics and the underlying pathways.O ur data indicates that the dialkyl-polyDNP conjugate was endocytosed, but at ar ate that is sufficiently slow to still allow for antibody recruitment to last over several days.This could be acrucial feature in vivo to allow for prolonged exposure of ARP-treated tumors to antibody recruitment and subsequent innate immune attack.…”

“…[21] Cholesterol-based conjugates have been reported to undergo fast exchange from the cell membrane,w hich might account for the less efficient antibody-recruiting capacity of cholesterol-based ARPs. [22] Using the dialkyl-polyDNP conjugate, we subsequently tested its persistence on the cell surface over prolonged periods of time by treating cells with polymers followed by pulsing with anti-DNP at different time points prior to flow cytometry and confocal microscopy analysis.I n these experiments,c ells were carefully washed to remove unbound polymer to avoid continuous replenishing of lipidpolymer on the cell surface from lipid-polymer in solution. These experiments ( Figure 2E)d emonstrate that for at least 4days post dialkyl-polyDNP treatment, anti-DNP can bind to the cell surface.W hile antibody-dependent effector mechanisms occur fairly rapidly (see below for ADCP data), we hypothesize that prolonged cell surface display in vivo of ARPs allows for ahigher chance of Fc-recognition to occur and innate effector killing to take place.M oreover, our data show that despite the fact that ARPs become endocytosed to ac ertain extent (as evidenced by the dotted red fluorescent intracellular pattern in the confocal images in Figure 2B), avery significant fraction of ARPs remains on the cell surface and is capable of recruiting anti-DNP to the cell surface for multiple days post pulsing of cells with ARPs.…”

Stability of Alkyl Chain-Mediated Lipid Anchoring in Liposomal Membranes

“…Previous research of our group established the monitoring of lipid escape from the liposomal membrane and insertion into other membranes. This behavior was particularly evident for cholesterol lipids, which, presumably due to their irregular scaffold structure, are less stably anchored in membranes than typical biomembrane lipids whose two long hydrocarbon chains are known to form a dense network of van-der-Waals interactions in a symmetric lipophilic environment such as a cell membrane [ 26 ]. In order to demonstrate this effect, we prepared liposomes based on functionalizable cholesterol and dialkyl lipid derivatives, to which fluorescent dyes were covalently attached by click chemistry.…”

Dual Centrifugation - A Novel “in-vial” Liposome Processing Technique

“…Beside the advantages of DC processing of many small sample vials in one run, the possibility to prepare very small batches significantly helps to save costs, e.g., when doing experiments with expensive materials such as siRNA [10,16], or when compounds to be entrapped or special lipids are only available in small amounts [35,37]. Thus, the typical batch sizes applied in the DC-homogenization are usually < 500 mg (2 ml vials), and batch sizes down to 20 mg [16] and 1-5 mg [37] have been reported as well.…”

“…Thus, the typical batch sizes applied in the DC-homogenization are usually < 500 mg (2 ml vials), and batch sizes down to 20 mg [16] and 1-5 mg [37] have been reported as well. These small batch sizes significantly differ from the minimal batch sizes when using HPH.…”