Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α‑Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44 Eliška Bartheldyová,† Roman Effenberg,‡ Josef Mašek,† Lubomír Procházka,† Pavlína Turánek Knötigová,† Pavel Kulich,† František Hubatka,† Kamila Velínská,† Jaroslava Zelníčková,† Darina Zouharová,† Martina Fojtíková,† Dominik Hrebík,§ Pavel Plevka,§ Robert Mikulík,# Andrew D. Miller,† Stuart Macaulay,¶ Daniel Zyka,∥ Ladislav Drož,∥ Milan Raška,*,†,⊥ Miroslav Ledvina,*,‡ and Jaroslav Turánek*,† †

Department of Pharmacology and Immunotherapy, Veterinary Research Institute, v.v.i., Hudcova 70, 621 00 Brno, Czech Republic Department of Chemistry of Natural Compounds, University of Chemistry and Technology, Technická 5, 166 28 Prague 6, Czech Republic § Central European Institute of Technology CEITEC, Structural Virology, Masaryk University, Kamenice 753/5, 62500 Brno, Czech Republic ∥ APIGENEX s.r.o., Poděbradská 173/5, Prague 9, 190 00, Czech Republic ⊥ Department of Immunology and Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hněvotínská 3, 775 15 Olomouc, Czech Republic # The International Clinical Research Center of St. Anne’s University Hospital Brno, 656 91 Brno, Czech Republic ¶ Malvern Instruments, Great Malvern WR14 1XZ, United Kingdom ‡

S Supporting Information *

ABSTRACT: New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling (Noxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

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INTRODUCTION Hyaluronic acid (HA) is a biodegradable, biocompatible, nontoxic, and non-immunogenic glycosaminoglycan used in a broad range of medicinal applications. HA is also a ligand for various receptors expressed on many types of cells including cancer cells. Reported interactions of HA with various cellular receptors, such as CD44,1,2 the receptor for hyaluronatemediated motility (RHAMM),3 the lymphatic vessel endothelial HA receptor (LYVE-1),4 and the HA receptor for endocytosis (HARE),5,6 make HA a promising candidate for © XXXX American Chemical Society

selective cellular delivery of imaging and anticancer agents and for immunotherapy, by exploiting opportunities for receptormediated active targeting offered by such receptor interactions.7 Therefore, modification of the surface of nanoparticles like liposomes with HA is of interest with respect to the development of new drug delivery platforms.8 Received: May 4, 2018 Revised: June 12, 2018 Published: June 13, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00311 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Synthetic route to aminoxylipids 8a, 8b, and 12. Detailed description is available in the Supporting Information.

Liposomes, self-assembly nanostructured vesicles, represent one of the most widely appreciated platforms for targeted drug delivery in vivo. Liposomes themselves are seen as nontoxic biocompatible nanoparticles approved by the Food and Drug Administration (FDA) for applications in human medicine.9 Liposomal products have already found a number of clinical applications, due to their biocompatibility, biodegradability, low toxicity, and ability to internalize substances with different physicochemical properties. The interior cavity as well as the envelope, consisting of a phospholipid bilayer, can be used to incorporate biologically active agents. In particular, the internal aqueous space can be used to encapsulate hydrophilic agents whereas the phospholipid bilayer can be used to anchor lipophilic agents via their hydrophobic domain(s).10 The latter approach has been used successfully for the construction of targeted liposomes, lipid-based microbubbles, and liposomal vaccines with bioactive agents mounted on liposome surfaces.11,12,21,13−20

Active and specific targeting of liposomal drug carriers is achieved through the modification of liposomal surfaces using various ligands including saccharides and polysaccharides. For this purpose, rapid and orthogonal reactions that take place under mild condition are needed for the production of such liposomes on both the laboratory and industrial scale. Two basic approaches have been applied in the past. One is based on the use of lipids modified by receptor-selective targeting molecule(s) as colipids in liposomal formulations. The alternative approach is based on a post-modification of already prepared liposomes with receptor-selective targeting molecule(s). In the second scenario the colipid used in the construction of liposomes bears a chemical coupling moiety that allows selective coupling of receptor-selective targeting molecule(s) onto liposomal surfaces.22,23 Lipids as components of a liposomal bilayer can be potentially attached to HA via the carboxylate group on the glucuronic acid residue, the primary hydroxyl on the Nacetylglucosamine moiety, or reductive amination chemistry B


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decided to replace acid 3 with its chloride and carry out the acylation in DCM, wherein the 2-tetradecylhexadecanoyl chloride (4) is soluble. The acyl chloride 4 was prepared by modification of the reported procedure27 (i.e., reaction of acid 3 with oxalyl chloride in the presence of a catalytic amount of DMF in DCM at room temperature, instead of reaction with thionyl chloride in benzene under reflux). The modified procedure provided acyl chloride 4 in analytical quality without any other purification, in comparison to the procedure described. N-Acylation of partially protected diamines 1a and 1b with chloride 4 in the presence DIPEA as base gave Nprotected acyl derivatives 5a and 5b in the significantly higher yield of 94% and 90%, respectively. The Boc protecting groups of N-Boc-protected lipoamine intermediates 5a, 5b, and 9 were removed by treatment with TFA in DMC at room temperature giving N1-(2-tetradecylhexadecanoyl)-1-amino-2-ammonioethane trifluoroacetate (6a), N1-(2-tetradecylhexadecanoyl)-1-amino-3-ammonio propane trifluoroacetate (6b), and N1-(2-tetradecylhexadecanoyl)-1amino-8-ammonio-3,6-dioxa-octane trifluoroacetate (10), respectively, that were used without any purification in the next step. HATU promoted condensation of N-Boc-aminoxy acetic acid 7 with trifluoroacetates of 6a, 6b, and 10 in the presence of NMM and catalytic amount of DMAP afforded N1-(2tetradecylhexadecanoyl)-N2-(N-tert-butoxycarbonylaminoxy acetyl)-1,2-diaminoethane (7a), N1-(2-tetradecylhexadecanoyl)-N3-(N-tert-butoxycarbonylaminoxy acetyl)-1,3-diaminopropane (7b), and N1-(2 tetradecylhexadecanoyl)-N8-(N-tertbutoxycarbonylaminoxy acetyl)-1,8-diamino-3,6-dioxa-octane (11) in the 64%, 76%, and 76% yields, respectively. By using DIC as a condensation agent in the presence of DIPEA and DCM as solvent, a higher conversion acylation was achieved to give compounds 7a, 7b, and 11 in the 86%, 88%, and 85% yield, respectively. These N-protected aminoxylipids were converted onto targets N1-(2-tetradecylhexadecanoyl)-N2ammoniooxy acetyl-1,2-diaminoethane trifluoroacetate (8a), N1-(2-tetradecylhexadecanoyl)-N3-ammoniooxy acetyl-1,3-diaminopropane trifluoroacetate (8b), and N1-(2 tetradecylhexadecanoyl)-N8-ammoniooxy acetyl-1,8-diamino-3,6-dioxa-octane trifluoroacetate (12) by Boc deprotections with TFA in DCM. Liposome Preparation and Characterization. Differences between the bindings of HA onto liposomes via carbodiimide (EDC) and via aminoxy coupling are shown (Figure 2). For experiments to evidence these differences we used nanoliposomes of about 70 nm with low polydispersity indexes (below 0.1) to reveal small changes in the size distribution owing to the binding of HA onto their surfaces. Binding of HA onto the surface of nanoliposomes using EDCmediated or aminoxy coupling was associated with an increase in liposome size and ζ-potential values in comparison to plain nanoliposomes (Figure 3). Binding of HA onto liposomes via EDC condensation was found to result in a slight increase in size (about 4 nm) and ζ-potentials similar to free HA (−21 mV). In contrast, binding of HA via aminoxy coupling was found to lead to a significant increase in nanoliposome size, and a drop in ζ-potential value to −8 mV in comparison with free HA (Table 1). Such data are consistent with the structural variations described (Figure 2). In the event that HA molecules are bound via EDC condensation, they should do so randomly using randomly positioned carboxylic groups, thereby resulting in the formation of a flat surface layer of HA molecules in random orientation. Corresponding ζ-potential

through the reducing end of HA. Liposomes, bearing HA conjugated to a phosphatidylethanolamine lipid, have been used for targeting CD44 receptors. This approach resulted in multipoint attachment of HA to liposomes.24 Aminoxy coupling reactions belong to the category of “click chemistry”16 and may be described as biorthogonal “click” ligations. This is because such reactions are based on the spontaneous condensation of aminoxy functional groups with an available aldehyde group or ketone to give rise to oxime link formation.25 These facts make the aminoxy coupling almost an ideal method for post-modification of liposomes with complex biologically active molecules. In the preparation of liposomes suited for post-liposomal oxime ligation, single- and doublestranded aminooxy lipids have been used. The hydrophobic domain of single-stranded aminooxy lipids is formed by linear hydrocarbon chains. In the case of aminoxy lipids with dual fatty acyl chains, the hydrophobic domain of these lipids is based on the symmetrical lipophilic di-O-acyl of glycerol.26 Cholesterol-based aminoxy lipids have also been described.22,23 Here we described syntheses of novel biocompatible aminoxy lipids suitable for incorporation into liposomes which were subsequently functionalized on the surface by hyaluronic acid via orthogonal aminoxy coupling. Here, we describe, for the first time, the application of microfluidics mixing techniques using the NanoAssemblr instrument for preparation of nanoliposomes with aminoxy lipids for postforming coupling of biomacromolecules containing aldehyde function. The support of the concept was demonstrated by massive internalization of HA-liposomes via CD44 receptor expressed on cancer cell lines.

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RESULTS Chemistry. The synthetic route to the target lipids presenting an aminoxy group 8a, 8b, and 12 is shown in Figure 1 (Figure 1). This synthetic scheme is based on the initial condensation of partially N-protected diamines 1a, 1b, and 2 differing in the length and hydrophobicity/hydrophilicity of spacers connecting amino groups, with 2-tetradecylhexadecanoic acid 3 in the presence of HATU as condensation agents or activated as its acyl chloride 4. This was followed by N-deprotection of acyl derivatives 5a, 5b, and 9 and subsequent N′-acylation of the free amines 6a, 6b, and 10 using N-Boc-aminoxyacetic acid 7 to give fully protected intermediates 7a, 7b, and 11. A final deprotection was then used to give rise to aminoxy lipids 8a, 8b, and 12. HATU promoted condensation of 2-tetradecylhexadecanoic (3) with N-Boc-1,2-diaminoethane hydrochloride (1a) or with N-Boc-1,3-diaminopropane hydrochloride (1b), in the presence of NMM and a catalytic amount of DMAP in DMF, afforded N1-(2-tetradecylhexadecanoyl)-N2-tert-butoxycarbonyl-1,2-diaminoethane (5a) and N1-(2-tetradecylhexadecanoyl)N3-tert-butoxycarbonyl-1,3-diaminopropane (5b) in 68% and 55% yield, respectively. On the other hand, following the same condensation of 2-tetradecylhexadecanoic (2) acid with NBoc-3,6-dioxa-1,8-diaminooctane (2), the N1-(2-tetradecylhexadecanoyl)-N8-tert-butoxycarbonyl-1,8-diamino-3,6-dioxa-octane (9) was obtained in significantly higher yield (90%). This discrepancy appeared caused by the observed limited solubility of the fatty acid 3 and hydrochlorides of the N-protected diamines 1a, 1b in DMF. By contrast, N-Boc-3,6-dioxa-1,8diaminooctane (2) is fully soluble in DMF. Consequently, we C


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structure layer comprising HA molecules in linear conformation orthogonal to the surface28 (Figure 2). In such a conformation, ionization of internal carboxyl groups can be expected to be suppressed, thereby reducing negative ζpotential values. HA can by linked onto liposomal surface via carbodiimide condensation involving amine groups of phospholipids (e.g., of phosphatidylethanol amine or else other amine functional groups linked to lipids via suitable spacers). This reaction results in HA bound to the liposomal surface in random orientations and the formation of a flat, highly negatively charged surface layer. Coupling of HA via aminoxy ligation ensures chemo- and regioselective coupling of HA to liposomal surfaces. Such coupling appears to lead to formation of a brush-like surface structure layer comprising HA molecules in linear conformation orthogonal to the liposome surface. Accordingly, most HA carboxylate groups are brought into close spatial relationship with each other leading to suppression of ionization (i.e., characterized by increased pKa values), hence lowering the negative ζ-potential value in comparison to free HA and HA-liposomes prepared by the carbodiimide condensation procedure. Secure covalent binding of HA onto liposome surfaces was confirmed by gel permeation chromatography separation on the column Superose 6 (Figure 4). Fluorochrome-labeled HA

Figure 2. Schematic presentation of orthogonal and random binding of HA onto liposomes.

Figure 4. Separation of HA liposomes by GPC. Plain or HA-coated nanoliposomes were separated by FPLC on the column Superose 6 (mobile phase: PBS, pH 7.2). HA used for coating of nanoliposomes was labeled with fluorescence probe rhodamine for detection. Fractions of 1 mL were collected and fluorescence signal was measured to quantify free HA and liposome-bound HA. 100 mM sodium citrate (pH 7.2) has been used to release noncovalently bound HA from the liposomal surface. Inset shows confocal microscopy image of fluorescent HA-liposomes eluted from GPC column.

Figure 3. Size distribution of HA, plain liposomes, and liposomes with surface HA via EDC condensation or aminoxy coupling. Liposomes were prepared by microfluidic mixing and extruded through polycarbonate filters (100 nm). The size distribution and ζpotential was measured by Zetasizer Nano ZS (ζ-potential was measured in 10 mM sodium phosphate pH 7.2 at 20 °C).

Table 1. Size, Polydispersity Index (PDI), and ζ-Potential of Plain and HA-Liposomes sample HA Plain Liposomes Liposomes + HA via EDC ligation Liposomes + HA via oxime ligation

Z-average (nm) 6.2 68.5 72.4 81.3

± ± ± ±

1 2 1 3

PDI 0.31 0.08 0.13 0.11

was coeluted with liposomes and only a small amount was eluted in the fraction corresponding to free HA. Incubation of HA-liposomes with 100 mM sodium citrate (pH 7.2) did not remove HA from liposomes as demonstrated by gel permeation chromatography separation. This confirms that majority of HA is not bound by electrostatic interactions, for example, ionic interactions between carboxylate functional groups of HA and quaternary amine functional groups of phosphatidylcholine in liposomes are unstable after incubation with carboxylic polyanions like citrate. In our case, only a marginal release of HA was demonstrated as a result of gel permeation chromatography (Figure 4). Electron Microscopy. Direct visualization of HA-layers at the surface of liposomes was performed using transmission electron microscopy (Figure 5). HA is readily stained by 2%

ζ-potential (mV) −21 +1 −20 −8

± ± ± ±

2 1 2 1

values should also remain unchanged from values of free HA. In comparison, aminoxy coupling has to be highly regioselective for the reducing terminus (hemiacetal) of HA, thereby enabling HA molecules to form a brush-like surface D


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Figure 5. Transmission electron microscope pictures of plain and HA-liposomes. (A) TEM of plain liposome, (B) HA-liposome, (C) mixture of plain and HA-liposome (black arrow indicates HA-liposomes, white arrow indicates plain liposome), (D) immunogold staining of HA-liposome (gold nanoparticles labeled with anti-HA antibody).

Figure 6. Cryo-TM of liposomes covered with HA after aminoxy coupling. Negatively charged HA was contrast-imaged by labeling with uranyl acetate (0.05% uranyl acetate). Positively charged UO2+2 binds HA selectively. An outer layer (more contrasted due to UO2+2 staining) was observed of about 4.5−6.7 nm that was taken to be the HA layer (black arrow). An inner layer was observed of about 3.7−4.2 nm that appears to be phospholipid bilayer (white arrow). The increase in liposome size from HA binding (as measured by DLS) was about 8−13 nm, which is in good correlation with these data from cryo-TM. DLS size: HA (5−15 kDa) Rh 2.5−3 nm (diameter 5−6 nm); Plain liposomes Rh 34 nm (diameter 68 nm); HA-liposomes Rh 40.5 nm (diameter 81 nm). Insetdetail of HA layer onto liposomal surface.

Interaction of HA-Liposomes with Cancer Cells Expressing CD44. Characterized HA-liposomes were tested for their ability to bind CD44 receptor under in vitro conditions. Cell lines selected for in vitro experiments were first tested for their overall ability to bind HA using standard FITC-labeled HA (data not shown). Various concentrations of the plain and HA-coated liposomes were incubated for 15 min and 24 h with nonsmall cell lung carcinoma cell line H1299 overexpressing HA receptor CD44. For comparison, human breast adenocarcinoma cell line T47D expressing very low levels of CD44 were used as a negative control for differential binding of HA-coated and plain liposomes (Figure 7C). Differential expression of CD44 in H1299 and T47D cells was evaluated using Western blot (Figure 7A). We also compared differential binding of HA-coated aminoxy liposomes to human cervical carcinoma HeLa cells before and after incubation with 50 nM TPA which induces overexpression of CD44 in HeLa cells (Figure 7B). Increase in CD44 expression in HeLa correlated with increased binding of HA-aminoxyliposomes to cells (Figure 7D). Flow cytometry confirmed that HA-

aqueous phosphotungstic acid and liposomal surface covered by HA is visualized as a rough structure. Structural identity of HA was confirmed by immunogold staining using HA-specific antibodies labeled with gold nanoparticles (Figure 5D). The layer of HA on the surface of liposomes was also visualized by cryo-transmission microscopy. In this case we used uranyl acetate as contrast agent with which to bind and identify negatively charged HA. HA layer is visualized as more contrast outer line with thickness of about 4.5−6.7 nm (Figure 6). The phospholipid bilayer is visualized as lighter inner line of about 3.7−4.2 nm. Cytotoxicity in Vitro Assay. Both MTT and flow cytometry assays demonstrated the in vitro biocompatibility of liposomal preparations. No cytotoxicity was detected in cells 24 and 48 h after incubation with liposomes composed of EPC, aminoxylipids, and HA-liposomes prepared by aminoxy coupling at a final total lipid concentration of ranging 1− 1000 μg/mL of cell culture. The percentage of apoptotic cells was the same as in nontreated control cells. Viability of cells was over 95%. E


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Figure 7. HA-liposomes target CD44 receptor. The level of CD44 protein expression in H1299 and T47D cells is shown in immunoblots using anti-CD44 IgG and anti-actin IgG as a loading control (A). 50 nM TPA upregulates expression of CD44 in HeLa cells in 18 h. Anti-GAPDH IgG was used as a loading control (B). Rhodamine-labeled liposomes were incubated with tested cell lines for 15 min and 24 h and observed with epifluorescence microscope (D). Quantification of HeLa cells binding FITC-labeled HA-liposomes by flow cytometry. HeLa cells were either pretreated (green line) or left untreated (red dashed line) with 50 nM TPA for 18 h prior to 15-min-long incubation with HA-aminoxyliposomes. HeLa cells without liposomes were used as a negative control (dotted line) (C). Plain liposomes represent EPC liposomes without aminoxy lipid and HA coat. HA-aminoxyliposomes represents HA coated liposomes containing aminoxy lipid. The HeLa cells analyzed by flow cytometry are those positive for CD44 after staining with mouse anti-CD44 IgG followed by FITC-labeled goat anti-mouse IgG. The Western blots and fluorescence images are representatives of at least three independent experiments.

modification of metastable structures like liposomes or proteoliposomes. Another advantage of the aminoxy coupling is a lack of any noxious byproducts as only water is produced. Therefore, purification of final product HA-modified liposomes is not necessary.31 Despite the fact that the oxime bond formed by aminoxy coupling is thermodynamically unstable and undergoes hydrolysis at an appreciable extent in aqueous solution under mildly acid conditions,22,23 this feature becomes a useful advantage in applications where controlled release based on defined slow hydrolysis of the covalent bond is needed. Tumor matrix or intracellular compartments like lysosome are good examples for controlled release applications.22,23,30,32 With respect to self-assembly nanoparticles like liposomes, lipids with two fatty acyl chains are structurally more similar to structural phospholipids. Therefore, they are better suited for incorporation into lipid bilayers than lipids with single fatty acyl chains. Single fatty acyl chain lipids anyway have a tendency to form micelles or reverse micelles that act to destabilize lipid bilayers.33−35 In this context, we considered it promising to use a fatty acid comprising a branched, single fatty acyl chain symmetrically branched at position C(2). Such an approach was used previously for the design and synthesis of new nontoxic polycationic lipids for the construction of polycationic liposomal carriers with applications in the field of anticancer/antiviral therapy, vaccinology, and gene therapy.36,37 In position C(2), symmetrically branched fatty acids having alkyl chains with even numbers of carbon atoms are biodegradable by β-oxidation as other biogenic fatty acids.38,39 Accordingly, we selected 2-tetradecylhexadecanoic branched at position C(2) as the single fatty acid from which the

aminoxyliposomes bound to TPA treated HeLa cells more intensively than to untreated control cells (Figure 7C).

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DISCUSSION Aminoxy Coupling. Most conventional procedures widely used for post-liposomal modifications are based on amide condensation, disulfide bridge formation, interconnection of two amino groups via homobifunctional linker, and addition of a sulfanyl group on the maleimide CC bond. A disadvantage of these reactions is their low chemoselectivity (orthogonality) in relation to other chemical functions present in complex biomolecules. Oxime functional groups are formed by the coupling of aminoxy groups to ketones or aldehydes. The reaction can be carried out in water and is optimal under mildly acidic conditions. Aminoxy compounds can react directly with unmodified hyaluronan.29,30 Oxime formation is a one-step alternative to two-step reductive aminations, e.g., formation of imine (Schiff bases) by reaction of amino group with aldehyde or keto group followed by reduction to amine. Oxime linkages with a Kd = 10−8 M are also much more resistant to hydrolysis than imines. Therefore, such oxime “click chemistry” is emerging as a robust strategy especially in the field of glycoconjugation, taking advantage of the fact that the reducing termini of most polysaccharides involve an aldehyde functional group in equilibrium with its hemiacetal form. Importantly, the resulting oxime bond is stable at physiological pH and thus suitable for drug delivery.22,23 Aminoxy coupling is highly chemo and regioselective so useful for the binding of polysaccharides like HA because only one reducing terminus (hemiacetal) is present in each HA molecule. Under aqueous conditions this reaction is useful for F


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microscopy (Figure 5), and cryo-transmission electron microscopy (Figure 6) demonstrated the efficient binding of HA to liposomes as suggested HA-liposome structures as well. Synthetic aminoxy lipids have proven applicable in the preparation of functionalized liposomes by classical methods like lipid film hydration and extrusion22,23 as well as with new methods based on microfluidic mixing. Microfluidic mixing represents promising technology for production of complex liposomal preparation by on-chip technology as demonstrated recently.45 Targeting of HA Liposomes to CD44 Expressing Cells. HA, as a component of extracellular matrix, plays an important role during all steps of the wound healing process. HA also plays a crucial role in healing processes and inflammation. It is especially concentrated in regions of cell division and invasion.46 Active role of HA in modulating tissue regeneration is based on interactions with various receptors located on external or internal surfaces of immune cells as well as on various types of endothelial cell, fibroblasts, and keratinocytes. RHAMM, CD44s, TLR-4, TL2-R, Stabilin-1 (HARE), and LYVE-1 are all known as key receptors triggering signaling pathways in response to interaction with various forms of HA.47,48 In general, low-molecular-weight HA triggers proinflammatory responses while high-molecular-weight hyaluronic acid triggers anti-inflammatory responses.49 The unique properties of HA enhanced some attempts to apply it in clinical practice, especially in drug targeting to CD44 expressing cancer cells, treatment of chronic wound and inflammation, as well as targeting of dendritic cells to enhance the efficacy of vaccines. Possible application of HA-targeted therapeutics is described in Figure 8. Therefore, binding of HA is an example of a polysaccharide having multiple physiological functions. CD44, main receptor for HA, is present on cell membranes of almost all human cells. Organization of CD44 gene into 10 constant and 10 variant exons gives a great number of different splicing variants. CD44 is responsible for cellular internalization of HA degradation products by macrophages. Various isoforms of CD44 receptor are expressed on cells of the immune system and on most tumor cells. Tumors are unique in proportion of expressed standard CD44 isoform (CD44s) and isoforms produced from splicing variants containing variable exons (CD44v).50 CD44 is appreciated also as a major receptor for HA on cancer stem cells of many tumors although its role in tumor progression still remains a subject of intensive research.50,51 Changes in CD44 and HA expression have been observed in many experimental tumors as well as in many animal models.52 It is believed that adhesion of tumor cells to extracellular matrix is one of the critical steps in tumorigenesis. HA-CD44 interaction can trigger a variety of intracellular downstream signaling pathways like MAP kinase and PI3 kinase-Akt and thus promote tumor cell proliferation, motility, chemoresistance, and survival.46,53−57 Low-molecular-weight hyaluronan, below 100 kDa, has proinflammatory and proliferative effects, while high-molecular-weight HA promotes transcriptional activation and differentiation.54 Ability of tumor cells to interact with HA through CD44 receptor is important for metastatic behavior of tumor cell xenografts in experimental animal models. This phenomenon was observed from the beginnings of CD44 research.55,57,58 HA-CD44 interactions are utilized in delivering of chemotherapeutic drugs into tumor cells.8,59 The anticancer effect of HA targeted drug delivery to cancer cells is related to CD44 expression status of cancer cells.60 We have

aminoxylipids reported here were constituted. Our studies on in vitro cell culture models confirms the biocompatibility of functionalized lipids based on C(2) symmetrically branched fatty acids. Orthogonality of HA coupling onto the liposomal surface can be achieved also by reaction of aldehyde group with amine presented in lipids like dipalmitoylphosphatidylethanolamine. In this case reductive amination is used to stabilize the linkage.40 Even if the reaction is well established and used in bioconjugation of HA and other polysaccharides, the applicability for preparation of liposomes by on-chip technology could be limited owing to the slow reaction rate. In this case, liposomes can by modified by HA also via postinsertion reactions. HA-lipid conjugate is prepared by suitable coupling reaction (e.g., carbodiimide condensation or reductive amination) and HA-lipid conjugate is added to preformed liposomes and inserted into the outer part of the lipid bilayer. Increasing the temperature above the Tm of the lipid bilayer significantly enhanced insertion of the HA-lipid conjugate.41 HA-lipid conjugates can form micelles, especially conjugates with low MW HA. This fact can slow down the process of insertion. If the process is not rapid enough, suitability to use this method for preparation of liposomes by microfluidic mixing and on-chip technology is limited. Considering future application of siRNA in gene therapy of cancer, nonviral vectors like liposomes targeted toward cancer cells expressing CD44 are tested.42,43 Microfluidization represents prospective technology for preparation of siRNA liposomes, and aminooxy ligation seems to be more suitable for post-forming modification of siRNA liposomes in comparison to carbodiimide condensation or reductive amination. Simplicity and velocity of the aminooxy ligation areaction, orthogonality, lack of byproducts, and compatibility with microfluidic mixing technology are the factors favoring aminooxy ligation. Chemoenzymatic synthetic route to preparation of HA conjugates represents another approach for preparation of HAphospholipid conjugates. In this case the high-molecularweight HA-lipid conjugate is prepared by carbodiimide condensation and consequently hyaluronidase modifies the conjugate to a low MW HA-lipid one. The semirandom fashion of high MW HA-lipid conjugate prepared by carbodiimide condensation restricts the digestion of the conjugate by the hyaluronidase so that a mixture of HA-lipid conjugates is formed encompassing a narrow range of moderately sized HA oligomers coupled with a lipid.44 Application of available low MW HA (e.g., available from CONTIPRO Company) and N-oxy lipids represents a suitable alternative to this approach. Application of Microfluidization for Preparation of Nanoliposomes and HA-Liposomes. The NanoAssemblr platform used in our study is a scalable, microfluidics-based system developed for preparation and manufacture of liposomes which uses custom engineered microfluidic cartridges to perform nanoprecipitation within milliseconds and in nanoliter-reaction volumes. This enables well-controlled preparation of liposomes with tunable sizes and narrow PDI values in a single step. Optimization of the parameters for production of liposomes by microfluidic mixing led to monodisperse liposomal preparations useful for precise measurement of binding of HA onto liposomes. Independent methods like dynamic light scattering (Figure 3) gel permeation chromatography (Figure 4), transmission electron G


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by HA. Selective orthogonal binding of HA onto the liposomal surface via aminoxy coupling has been demonstrated and is compatible with the modern method for preparation of nanoliposomes by microfluidic mixing. In this study liposomes with orthogonally bound HA were proven to bind selectively to cells positive for CD44, such as HT1299 and CD44+ HeLa, but not to cells negative for CD44, such as T47D and CD44− HeLa. This is an important observation for applicability of HAliposomes as targeting particulate carriers for drug or vaccine delivery. Therefore, our novel aminoxy lipids have proven themselves as useful chemical tools for development and preparation of various liposomal platforms for targeted drug delivery. The liposomal platform is suitable not only for coupling of HA of various molecular weights, but also for other oligo- and polysaccharides with free reducing termini like mannan (manuscript submitted to Chem. Sci.). Postmodification of liposomes is of interest also for industrial production because there are no byproducts of the coupling reaction that need to be removed by separation methods.

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Figure 8. Schematic presentation of possible application of HAliposomes in medicine. Liposomes modified on their surface by HA can be targeted to various cells expressing specific receptors. Cancer cells expressing CD44v can be targeted by HA-liposomes carrying anticancer drugs. Selective targeting to CD44v positive cancer cells could be achieved by enhanced CD44v affinity for HA after homomolecular association of CD44v by specific antibodies. The other HA receptor on cancer cells is RHAMM, which is expressed also on dendritic cells. Besides RHAMM, dendritic cells express CD44s, Stabilin 1, LYVE-1, TLR-4, and TLR-2 which could contribute to enhanced immunogenicity of HA-liposomes carrying antigens and molecular adjuvants for vaccination purposes. HA liposomes could also target liver sinusoidal endothelial cells (LSEC) through LYVE-1 and stabilins. Targeting the HA-proteoliposomes to LSEC could be employed for induction of T cell tolerance.

EXPERIMENTAL PROCEDURES Chemicals. All chemicals, unless otherwise specified, were purchased from Sigma (St. Louis, MO). Synthesis. 1H and 13C NMR spectra were measured on a Bruker AVANCE instruments-500, Bruker AVANCE-400, and Varian UNITY INOVA 400 (1H at 500, 400 MHz, 13C at 125, and 100 MHz). Chemical shifts (δ) of protons and carbons are given in units of ppm and coupling constants (J) in Hz. Spectra were measured in deuterated solvents (CDCl3, CD3OD, DMSO-d6, (CD3)2CO, and D2O) as an internal standard TMS has been used or the central peak of solvent. IR spectra were measured in solution (CHCl3) by Bruker IFS-55. ESI MS were measured on the instrument LCQ Classic (Thermo Finnigan), HR-ESI MS on a Q-TOF micro (Waters). Elemental analyses (C, H, N) were performed on the PE 2400 Series II CHNS/O Analyzer (PerkinElmer, Waltham, Massachusetts, USA), determination of Cl, Br, and S titration methods. The course of reactions and product purity were monitored by TLC chromatography on Merck aluminum sheet silica gel 60 F254 and the substances were detected with UV light (254 nm) or 10% H2SO4 in methanol, followed by heating. Column preparative chromatography was performed on silica gel Fluka silica gel flash chromatography, for 60 (230−400 mesh). Chemicals used in the experiments were commercially available in the state, without further purification. Three types of lipids were synthesized8a, 8b, and 12. All three lipids were found to behave interchangeably so we use the term aminoxy lipid for all, since this is the key characteristic for all of these lipids. Therefore, for simplicity, only data using aminoxy lipid 12 are shown below on the basis that data obtained with aminoxy lipids 8a and 8b were found equivalent (Figure 9). 2-Tetradecylhexadecanoyl Chloride (4). To a stirred solution of 2-tetradecylhexadecanoic acid (3) (1.5 g; 3.32 mmol) in DCM (50 mL) oxalyl chloride (0.42 g; 6.63 mmol) and a catalytic amount of DMF were added. The reaction mixture was stirred at room temperature for 4 h and the solvent was distilled off in vacuo. The residue was dissolved in benzene (100 mL) and solution was washed with saturated solution of NaHCO3 (2 × 50 mL) and H2O (2 × 50 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The residue after co-distillation with DCM (3 × 3 mL) gave 1.52 g (97%) of crystalline acyl

utilized this CD44-HA interaction in our experiments with prototypic HA coated liposomal nanoparticles where HA is bound via aminoxy coupling to the membrane structural lipid. Significant differences in the uptake rate of plain and HAliposomes by CD44 positive cancer cells demonstrated the usability of the whole platform for further development of targeted anticancer drugs formulated in HA-nanoliposomes.61 HA is not only a CD44 receptor targeting molecule. For instance, surface modification of liposomes with HA presents a strategy to endow liposomes with stealth properties in circulation without inducing accelerated blood clearance and the triggering of hypersensitivity reactions in certain sensitive individuals owing to complement activation after repeated administration.62 Thus, HA, as an alternative to polyethylene glycol modification, represents an effective and promising strategy for the design and construction of future drug delivery systems that may greatly increase the utility of liposomes as drug delivery vehicles with prolonged circulation in vivo. Moreover, HA anchored at the surface of unilamellar liposomes clearly acts as a built-in cryoprotectant. This HA ability is not restricted to a specific drug, specific liposome formulation, or size range.63 This unique feature is suitable for on-chip technologies reducing problems with additional pharmaceutical cryoprotectants and adjustment of final liposomal preparations.

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CONCLUSIONS New synthetic lipids with aminoxy functional groups were synthesized for application in post-modification of liposomes H


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63% ethyl acetate, 14 mL/min, 64 min; the samples were applied to the column in CHCl3). The homogeneous fraction was evaporated in vacuo and the obtained residue was lyophilized from 1,4-dioxane. For 1a: The reaction gave 255 mg (68%) of 5a, as a white lyophilizate. For 1b: The reaction gave 210 mg (55%) of 5b, as a white lyophilizate. Method A for 2. 2-Tetradecylhexadecanoic acid (3; 300 mg; 0.66 mmol), HATU (278 mg; 0.73 mmol), and DMAP (catalytic amount) were dried in an apparatus equipped with a septum at room temperature and 20 Pa for 4 h. The apparatus was flushed with Ar (2×) and then dry DMF (11 mL) and NMM (134 μL) were added through the septum. The reaction mixture was stirred 1 h at room temperature and then the solution of 1-(t-butyloxycarbonylamino)-3,6-dioxa-8-octamine (2; 156 mg; 0.63 mmol) in dry DCM (10 mL) was added through the septum and stirring was continued overnight. Solvents were evaporated in vacuo and the residue was dissolved in DCM (15 mL) and obtained solution was washed with sat. aq. NaHCO3 (2 × 20 mL), 5% aq. NaHSO4 (2 × 20 mL), and H2O (2 × 20 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was codistilled with 1,4-dioxane (3 × 10 mL). The crude product was purified by flash chromatography on silica gel column (120 mL) in toluene-ethyl acetate (gradient 0−50% ethyl acetate, 14 mL/min, 84 min, the sample was applied to the column in CHCl3). The homogeneous fraction was evaporated in vacuo and the obtained residue was lyophilized from 1,4-dioxane to give 387 mg (90%) of 9 as a white lyophilizate. Method B. To a stirred solution of 2-tetradecylhexadecanoyl chloride (4; 0.39 g; 0.82 mmol) in DCM (25 mL) N-tertbutoxycarbonyl-1,2-diaminoethane hydrochloride (1a; 174 mg; 0.88 mmol) or N-tert-butoxycarbonyl-1,2-diaminopropane hydrochloride (1b; 186 mg; 0.88 mmol) or 1-(t-butyloxycarbonyl-amino)-3,6-dioxa-8-octamine (2; 218 mg; 0.88 mmol) and DIPEA (240 μL) were added and the mixture was stirred at room temperature overnight. Then, the mixture was diluted with DCM (50 mL) and the obtained solution was washed with 5% aq. NaHSO4 (2 × 15 mL) and H2O (2 × 15 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude products were purified by flash chromatography on silica gel column (110 mL) in toluene-ethyl acetate. For 1a and 1b: gradient 0−63% ethyl acetate, 14 mL/min, 64 min; for 2: gradient 0−84% ethyl acetate, 15 mL/min, 44 min. The samples were applied to the column in CHCl3. Homogenous fractions of the products were evaporated in vacuo and finally lyophilized from 1,4-dioxane. For 1a: The reaction gave 454 mg (93%) of 5a, as a white lyophilizate. For 1b: The reaction gave 460 mg (94%) of 5b, as a white lyophilizate. For 2: The reaction gave 504 mg (90%) of 9, as a white lyophilizate. N 1 -(2-Tetradecylhexadecanoyl)-1-amino-2-ammonioethane trifluoroacetate (6a), N1-(2-tetradecylhexa-decanoyl)-1-amino-3-ammoniopropane trifluoroacetate (6b), N1(2-tetradecylhexadecanoyl)-1-amino-8-ammonio-3,6dioxa-octane trifluoroacetate (10). N1-(2-Tetradecylhexadecanoyl)-N2-tert-butoxycarbonyl-1,2-diaminoethane (5a; 420 mg; 0.71 mmol) or N1-(2-tetradecylhexadecanoyl)-N3-tertbutoxycarbonyl-1,3-diaminopropane (5b; 430 mg; 0.71 mmol), or N1-(2-tetradecylhexadecanoyl)-N8-tert-butoxycar-

Figure 9. Reagents and conditions: (a) 3, [CH3(CH2)13]2CHCOOH, HATU, DMAP, NMM, in DMF, r.t., 68% for 5a, 55% for 5b, 90% for 9; (b) 4, [CH3(CH2)13]2CHCOCl, DIEA, in DCM, r.t., 93% for 5a, 94% for 5b, 90% for 9; (c) TFA, in DCM, r.t.; (d) HATU, DMAP,NMM, in DMF, r.t., 64% for 8a, 76% for 8b, 76% for 11; (e) DIC, DMAP, in DCM, r.t., 86% for 7a, 88% for 7b, 85% for 11; (f) oxalyl chloride, DMF, in DCM, r.t.

chloride (4); mp 51−52 °C. Ref. 20, mp 51−53 °C. For C30H59ClO (471.24) calculated: 76.46% C, 12.62% H, 7.52% Cl; found: 76.28% C, 12.60% H, 7.32% Cl. N1-(2-Tetradecylhexadecanoyl)-N2-tert-butoxycarbonyl1,2-diaminoethane (5a), N1-(2-tetradecylhexa-decanoyl)N3-tert-butoxycarbonyl-1,3-diaminopropane (5b). N1-(2-tetradecylhexadecanoyl)-N8-tert-butoxycarbonyl-1,8-diamino3,6-dioxa-octane (9). Method A for 5a and 5b. 2Tetradecylhexadecanoic acid (3; 300 mg; 0.66 mmol), Ntert-butoxycarbonyl-1,2-diaminoethane hydrochloride (1a; 124 mg; 0.63 mmol) or N-tert-butoxycarbonyl-1,3-diaminopropane hydrochloride (1b; 132 mg; 0.63 mmol), HATU (278 mg; 0.73 mmol), DMAP (catalytic amount) were dried in an apparatus equipped with a septum at room temperature and 20 Pa for 4 h. The apparatus was flushed with Ar (2×), and then dry DMF (11 mL) and NMM (134 μL) were added through the septum. The mixture was stirred 1 h at room temperature, and then dry DCM (20 mL) was added through the septum and stirring was continued overnight. Solvents were evaporated in vacuo and the residue was dissolved in DCM (15 mL) and washed with sat. aq. NaHCO3 (2 × 20 mL), 5% aq. NaHSO4 (2 × 20 mL), and H2O (2 × 20 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was codistilled with 1,4-dioxane (3 × 10 mL) and the crude product was purified by flash chromatography on silica gel column (120 mL) in toluene-ethyl acetate (gradient 0− I


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the products were evaporated in vacuo and finally lyophilized from 1,4-dioxane. For 6a: The reaction gave 125 mg (86%) of 7a, as a white lyophilizate. For 6b: The reaction gave 132 mg (88%) of 7b, as a white lyophilizate. For 10: The reaction gave 142 mg (85%) of 11, as a white lyophilizate. N1-(2-Tetradecylhexadecanoyl)-N2-ammoniooxy acetyl1,2-diaminoethane trifluoroacetate (8a), N1-(2-tetradecylhexadecanoyl)-N3-ammoniooxy acetyl-1,3-diaminopropane trifluoroacetate (8b), N1-(2 tetradecyl hexadecanoyl)-N8ammoniooxy acetyl-1,8-diamino-3,6-dioxa-octane trifluoroacetate (12). N1-(2 Tetradecylhexadecanoyl)-N2-(N-tertbutoxycarbonylaminoxy acetyl)-1,2-diaminoethane (7a; 200 mg; 0.3 mmol) or N1-(2 tetradecylhexadecanoyl)-N3-(N-tertbutoxycarbonylaminoxy acetyl)-1,3-diaminopropane (7b; 205 mg; 0.3 mmol) or N1-(2 tetradecylhexadecanoyl)-N8-(N-tertbutoxycarbonylammoniooxy acetyl)-1,8-diamino-3,6-dioxa-octane (11; 223 mg; 0.3 mmol) were stirred in a mixture of DCM (8 mL) and TFA (8 mL) at room temperature for 2.5 h. The solvents were distilled off in vacuo. The residue was codistilled with DCM (3 × 4 mL) and the residue was lyophilized from 1,4-dioxane, to afford 8a or 8b or 12 in quantitative yields, as a white lyophilizates. Complete NMR and MS spectra are presented in the section Supporting Information Liposome Preparation. Microfluidic Mixing. Liposomes were prepared using a method based on microfluidic mixing. Mixture of lipids (EPC and aminoxy lipid 95:5 molar %) was dissolved in ethanol and then it was injected by disposable syringes into appropriate buffer using NanoAssemblr instrument (Precision Nanosystems, Vancouver, Canada). Ratio of water:ethanol phase was 7:1. The flow rate was 6 mL/min. Fluorescent liposomes contained 0.4 mol % of LissamineRhodamine phosphatidyl ethanolamine. Preparation of HA Liposomes by Aminooxy Ligations. Liposomes (10 mg/mL) with aminoxy lipid (1 mol % of total lipid) were mixed with HA (Mr 10440, concentration 1 mg/ mL). The ratio between total lipid and HA was 1 mg lipid: 0.1 mg HA. Incubation took place at room temperature for at least 30 min. Redundant unbound HA was removed by FPLC, column Superose 6 (Pharmacia, Stockholm, Sweden).64 Preparation of HA Liposomes by Carbodiimide (EDC) Condensation. HA was activated by EDC in molar ratio 1:1. Activation was carried out at room temperature for 2 h. Redundant EDC was removed by dialysis (Dialysis tubes − Merck Milipore, Billerica, Massachusetts, USA). Activated HA was mixed with liposomes containing 1% DOPE. Incubation was carried out at room temperature for at least 30 min. Redundant unbound HA was removed by FPLC, column Superose 6.64 Preparing of HA-Aminoxyliposomes Labeled by Gold Particles. For labeling by gold nanoparticles, liposomes modified by HA were used. Anti-HA antibody was then added and the whole incubated for 1 h. The ratio between HA and antibody was 0.2 mg HA to 1 mg antibody. Protein Abound gold nanoparticles (size 15 nm) were diluted 1:20 with PBS. Thereafter, 10 μL of diluted gold nanoparticles was added to 300 μL of the HA-aminoxyliposomes/anti-HA antibody sample. Nonbound molecules were removed by FPLC, column Superose 6. HA-liposomes covered by gold nanoparticles were observed by TEM.

bonyl-1,8-diamino-3,6-dioxa-octane (9; 485 mg; 0.71 mmol) were stirred in a mixture of DCM (12 mL) and TFA (1.5 mL) at room temperature for 3 h. The solvents were distilled off in vacuo. The residue was codistilled with DCM (3 × 3 mL) and subsequently lyophilized from 1,4-dioxane, to afforded 6a, 6b and 10 in quantitative yields, as a white lyophilizates. N1-(2-Tetradecylhexadecanoyl)-N2-(N-tert-butoxycarbonylaminoxy acetyl)-1,2-diaminoethane (7a), N1-(2-tetradecylhexadecanoyl)-N3-(N-tert-butoxycarbonylaminoxy acetyl)-1,3-diaminopropane (7b), N1-(2 tetradecylhexadecanoyl)-N8-(N-tert-butoxycarbonylaminoxy acetyl)-1,8-diamino3,6-dioxa-octane (11). Method A. N1-(2-Tetradecylhexadecanoyl)-1-amino-2-ammonioethane trifluoroacetate (6a; 490 mg; 0.81 mmol) or N1-(2-tetradecylhexadecanoyl)-1-amino-3ammoniopropane trifluoroacetate (6b; 500 mg; 0.81 mmol) or N1-(2-tetradecylhexadecanoyl)-1-amino-8-ammonio-3,6dioxa-octane trifluoroacetate (10; 624 mg; 0.81 mmol), Ntert-butoxycarbonylaminoxy acetic acid (7; 153 mg; 0.8 mmol), HATU (304 mg; 0.8 mmol), and DMAP (in catalytic amount) were dried in an apparatus equipped with a septum at room temperature and 20 Pa for 4 h. The apparatus was flushed with Ar (2×) then dry DMF (15 mL) and NMM (2 mL) were added through the septum. The mixture was stirred 20 min at room temperature, then dry DMF was added through the septum until complete dissolution of the reaction mixture (15 mL) and the stirring was continued overnight. Solvents were evaporated in vacuo and the residue was dissolved in DCM (40 mL) and washed with sat. aq. NaHCO3 (2 × 25 mL), 5% aq. NaHSO4, (2 × 25 mL), and H2O (2 × 25 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo and the residue was codistilled with 1,4dioxane (3 × 20 mL). The crude products were purified by flash chromatography on silica gel column (200 mL) in toluene-ethyl acetate. For 6a and 6b: gradient 0−84% ethyl acetate, 14 mL/min, 64 min; for 10: gradient 0−100% ethyl acetate, 14 mL/min, 43 min. The homogeneous fractions of the products were evaporated in vacuo and finally lyophilized from 1,4-dioxane. For 6a: The reaction gave 345 mg (64%) of 7a, as a white lyophilizate. For 6b: The reaction gave 410 mg (76%) of 7b, as a white lyophilizate. For 10: The reaction gave 465 mg (76%) of 11, as a white lyophilizate. Method B. To a stirred solution of N1-(2-tetradecylhexadecanoyl)-1-amino-2-ammonioethane trifluoroacetate (6a; 135 mg; 0.22 mmol) or N1-(2-tetradecylhexadecanoyl)-1amino-3-ammoniopropane trifluoroacetate (6b; 137 mg; 0.22 mmol) or N1-(2-tetradecylhexadecanoyl)-1-amino-8-ammonio-3,6-dioxa-octane trifluoroacetate (10; 158 mg; 0.22 mmol) in DCM (10 mL) DMAP (38 mg; 0.31 mmol), Ntert-Butoxycarbonylaminoxy acetic acid (7; 58 mg; 0.27 mmol) and DIC (41 μL; 0.26 mmol) were added and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (15 mL) and the obtained solution was washed with 5% aq. NaHSO4 (2 × 10 mL) and H2O (2 × 10 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude products were purified by flash chromatography on silica gel column (100 mL) in toluene-ethyl acetate. For 6a and 6b: gradient 10−73% ethyl acetate, 14 mL/min, 74 min; for 10: gradient 10−100% ethyl acetate, 14 mL/min, 74 min. The homogeneous fractions of J


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in 200 μL of PBS. FSC/SSC dot blot was used to gate the cells and exclude cellular debris or HA-liposomes. Fluorescence intensity of the FITC-label cells was measured using FL1 channel and expressed as histogram. Epifluorescence Microscopy. Immunofluorescence images were captured using the inverted epifluorescence microscope T 200 equipped with a digital camera (CCD1300, Nikon, Tokyo, Japan) and the LUCIA software (LIM, Czech Republic). Confocal Scanning Laser Microscopy. Confocal scanning laser microscopy was used for the observation of FITClabeled HA (FITC-HA) binding onto the surface of aminoxyliposomes. Liposomes (size of 400 nm) for the CSLM study were prepared according to the method of lipid film hydration followed by a thawing−freezing step and subsequent extrusion through the filter of 400 nm pore size. The lipid composition was the same as used for the preparation of the monodisperse liposomes by microfluidic mixing as described above. Liposomes were modified by FITC-HA (0.21 mg FITC-HA/1 mg lipid), separated from unbound FITC-HA by Superose 6 column, and immobilized onto the surface of chitosan nanofibers.19 The sample was observed by a confocal scanning microscope SP-2 (Leica, Wetzlar, Germany) at the following parameters: excitation laser 488 nm and optoacoustic filter set at emission spectrum of 505−530 nm. FPLC. Liposomes and unbound HA were separated by FPLC. Chromatographic condition: column Superose 6; mobile phase PBS pH 7.2; sample volume was 200 μL; flow rate was set at 0.1 mL/min at sample application and for separation the flow rate was set at 0.3 mL/min; volume of collected fractions was 1 mL. The fractions were analyzed with fluorescence spectrometer (PerkinElmer, Waltham, Massachusetts, USA). Cytotoxicity in Vitro Assays. Cytotoxicity of individual lipids and liposome preparations was tested using HeLa cells, A549 cells, and H1299 cells exposed for 48 h to liposomes composed of EPC, aminoxy lipids, and HA-liposomes prepared by aminoxy coupling at final concentration of total lipid ranging 1−1000 μg/mL of total cell culture volume. Cells (1.5−5 × 105) were seeded in 6-well plates in total volume 1 mL of complete growth medium (RPMI 1640 + 10% FCS). 200 μL of tested samples were added and cells were incubated for 24 h in 37 °C. Apoptotic cells were stained by Annexin V according to manufacturer’s recommendation (Invitrogen) and quantified by flow cytometer Fortessa (Beckton Dickinson, Franklin Lakes, New Jersey, USA). The percentage of apoptotic cells was measured also by use of the MTT test. Cells (1.5−5 × 104) were seeded in 98-well plates in total 100 μL of complete growth medium. 20 μL of tested sample was added and the cells were incubated for 24 h at 37 °C. Then, 20 μL of MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, 2,5 mg/mL PBS) was added. After 24 h, 10% SDS was added to dissolve MTT crystal. Finally, the absorbance was measured at 540 nm using a plate reader (Synergy II, BioTek, Winooski, Vermont, USA). Measurement of the Size of Liposomes and HA. The size and ζ-potential of HA and liposomes were measured by Zetasizer Nano ZSP (Malvern, UK). The size was measured at 25 °C in PBS (quartz cuvette 12 μL), concentration of lipid was 5 mg/mL, concentration of free HA 1 mg/mL. ζ-Potential was measured at 25 °C in 10 mM sodium phosphate buffer pH 7.2 (DTS 1060 cuvette). Concentration of liposomes was 50 μg of total lipid; concentration of free HA was 10 μg/mL.

Electron Microscopy. Samples of liposomes were suspended within a drop of PBS. The resulting suspension was covered with a grid coated with Formvar film (SigmaAldrich, Czech Republic) and carbon (Agar Scientific, Austria). The grid was removed from the suspension after 1 min, and the residual water was dried with a strip of filter paper. A drop of 2% aqueous phospho-tungstic acid was placed onto the grid for a few seconds, then excess stain was dried with filtration paper. Samples were observed under an electron microscope Philips 208 S Morgagni (FEI, Czech Republic) at 7500× magnification and an accelerating voltage of 80 kV. Cryo-EM Sample Preparation and Micrograph Acquisition. Prior to sample vitrification, quantifoil grids (R2/1, mesh 300 grid) were cleaned in plasma cleaner (Quorum Technologies) by exposure to plasma for 15 s. The samples for cryo-EM analyses were prepared by applying 3.8 μL of liposome suspension at a concentration of 1 mg/mL onto a grid, blotted, and vitrified by rapid plunging into liquid ethane using a Vitrobot Mark IV (FEI) that was operated at 20 °C, 100% humidity, blot time 2 s, blot force −2. The grids were then stored in liquid nitrogen. Micrographs were acquired using a Tecnai F20 (FEI) microscope operated at 200 kV, equipped with cryo-holder (Gatan, Inc.) and Eagle CCD camera (FEI). The microscope was aligned to achieve parallel electron beam and dose was calibrated to ∼22 e−/(Å2 s). Nominal defocus was set to ∼3.5 μm. Cell Culture and Treatment. The p53-deficient H1299 nonsmall cell lung cancer cell line and ductal carcinoma cell line T47D was grown in RPMI-1640 medium supplemented with antibiotics and 10% FBS. HeLa cells were cultured in EMEM medium supplemented with antibiotics and 10% FBS. The cells were treated at 80−90% confluence. Reagents and Antibodies. Mouse anti-actin IgG (AC-15 clone) monoclonal antibody was used at final dilution 1:10 000; rabbit anti-GAPDH polyclonal antibody was used at final dilution 1:5000; anti-pan CD44 IgG(2C5 BBA10, R&D Systems) was used at final dilution 1:1000, FITC-labeled goat anti-mouse IgG antibody (4408S, Cell Signaling) was used at final dilution 1:100, horseradish peroxidase-labeled anti-mouse IgG-HRP (A4416) was used at final dilution 1:1500, antirabbit IgG-HRP (NA934VS, GE Healthcare) was used at final dilution 1:1000. Western Blot Analysis. To obtain whole cell lysates, the cells were spun down at 200g for 3 min and washed two times with ice-cold PBS. The cell pellet was resuspended in WCL (whole cell lysis buffer) containing 10 mM Tris at pH 7,4, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 0.1%SDS, 1% Triton X-100, and protease inhibitor cocktail, frozen, defrosted, and sonicated as described.65 Aliquots were used to estimate a protein level using BCA method (Pierce) and the rest was diluted in 2×Laemmli loading buffer and boiled for 4 min. Proteins were separated by SDS/PAGE and transferred to PVDF membrane (Bio-Rad). After probing with a specific primary antibody and a horseradish peroxidase-conjugated secondary Ab, the protein bands were detected with enhanced chemiluminiscence kit (Pierce) using X-ray medical film (Kodak). Flow Cytometry. Liposomal targeting of HeLa cells was evaluated by flow cytometry (FACS Calibur, BD Bioscience, Franklin Lakes, New Jersey, USA) using the FITC-labeled HA liposomes. HeLa cells were detached and resuspended in FBSfree culture medium prior to incubation with liposomes. After incubation, cells were washed twice with PBS and resuspended K


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ASSOCIATED CONTENT

DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; FDA, Food and Drug administration; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; HA, hyaluronic acid; HARE, hyaluronic acid receptor for endocytosis; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HeLa, immortal cell line from cervical cancer from Henrietta Lacks; IgG, imunoglobulin G; LYVE-1, lymphatic vessel endothelial hyaluronan receptor; MTT, 3[4,5-dimetylthiazol-2-yl]-2,5-difenyltetrazolium bromid; NMM, N-methylmorpholine; PDI, polydispersity index; RHAMM, hyaluronan-mediated motility receptor; T47D, epihelial cells from human breast tumor; TEM, transmission electron microscopy; TFA, trifluoroacetate; TLR-2, toll-like receptor 2; TLR-4, toll-like receptor 4; TPA, 12-Otetradecanoylphorbol 13-acetate

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00311. Complete NMR and MS spectra of prepared lipids (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], Phone: +420 533 331 311. *E-mail: [email protected]. Phone: +420 220444283. *E-mail: [email protected]. ORCID

Jaroslav Turánek: 0000-0001-8001-4047

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Author Contributions

The contribution of the two first authors was equal. The team responsible for design and synthetic work is led by Dr. M. Ledvina, Dr. L. Drož, Ass. Prof. J. Turánek. The teams responsible for formulation of liposomes is led by Ass. Prof. J. Turánek; team responsible for testing of biological activities in vitro is led by Ass. Prof. J. Turánek, Prof. M. Raška, and ass. ́ The rest of the team contributed as prof. Robert Mikulik. follows: Roman Effenberg − synthesis and purification of precursors and final products. Eliška Bartheldyová, Josef ́ Mašek, Stuart Macaulay František Hubatka, Kamila Velinská , ́ Jaroslava Zelnič́ ková, Darina Zouharová, Martina Fojtiková − Preparation and characterization of liposomes, development of procedure and preparation of liposomes by microfluidization. ́ Pavel Plevka, and Pavel Kulich, Josef Mašek, Dominik Hrebik, Jaroslav Turánek − SEM, TEM, and cryo-TEM microscopy. ́ Turánek-Knötigová, Josef Mašek, Lubomiŕ Procházka Pavlina − in vitro testing, fluorescence microscopy testing, and CD44 targeting.

REFERENCES

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the following grants: the Ministry of Education, Youth and Sports OPVVV PO1 project “FIT” (Pharmacology, Immunotherapy, nanoToxicology) CZ.02.1.01/0.0/0.0/15_003/0000495 (JT) and LO1304 (MR); Project Centre of Excellence for Nanotoxicology CENATOX GBP503/12/G147 (J.T.); the Ministry of Health CZ AZV-Č R 15-32198A (M.R., J.T.); and CZ AZV-Č R 1630299A (J.T., J.M., and R.M.); the project MZE0002716202 of the Czech Ministry of Agriculture (J.T.). Grant Agency of the Czech Republic grant agreement 15-21631Y and from EMBO grant agreement EMBO-IG #3041 (P.P.) Access to instruments in the laboratory NanoPharm (joint project of Veterinary Research Institute and International Clinical Research Center (FNUSA-ICRC) is acknowledged, as well as support by Education and Research Centre VRI-Malvern. Authors are obliged to prof. Andrew D. Miller and ing. ́ Ludmila Faldiková for careful reading of the manuscript and language corrections.

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ABBREVIATIONS USED CD44, cluster of differentation 44; CD44s, standard isoform of CD44; CD44v, variable exon-encoded isoform of CD44; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; L


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Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal

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