Liposomes Modified with Superhydrophilic Polymer Linked to a Nonphospholipid Anchor Exhibit Reduced Complement Activation and Enhanced Circulation

Abstract

We report the synthesis of an acyl-anchored superhydrophilic polymer (SHP) for external surface modification of liposome surface. N¹-(2-aminoethyl)-N⁴-hexadecyl-2-tetradecylsuccinamide conjugated with SHP (HDAS–SHP) was synthesized and used for modifying the liposome surface. Unlike polyethylene glycol (PEG)–phospholipids, which are commonly used for manufacturing stealth liposomes, HDAS–SHP is devoid of both PEG and phosphoryl groups and possesses a zwitterionic polymeric chain. Circulation persistence of the ^99mTc-labeled HDAS–SHP liposomes was documented by gamma camera imaging. After 24 h postinjection, approximately 30% of injected HDAS–SHP liposomes were present in blood as compared with only 4.5% of the plain liposomes. HDAS–SHP liposomes inhibited complement activation. They were found to be amenable to pH-gradient-based active loading of Adriamycin in a stable manner. At 37°C, HDAS–SHP liposomes provided better encapsulation efficiencies than the liposomes modified with DSPE-PEG₂₀₀₀. These results provide a strong basis for HDAS–SHP as a viable alternative to PEG–phospholipids for imparting stealth characteristics to drug delivery vehicles such as liposomes.

INTRODUCTION

Liposomes have negotiated many delivery and toxicity-related challenges, especially for anticancer drugs.¹ Once encapsulated, the drugs’ distribution and clearance is determined by the pharmacokinetics of the liposomes. The pharmacokinetics of conventional liposomes is strongly affected by their encounter with complement proteins (C3, C4, and C5, laminin, fibronectin, C-reactive protein, type I collagen, etc.). Liposomes opsonized by these proteins are rapidly cleared from circulation via the mononuclear phagocyte system (MPS).^2–5 As such, prolonging circulation persistence of liposomes is a desirable goal in formulation development process. The opsonization can be minimized by modifying the liposomal surface with biocompatible natural or synthetic materials.^4,6–9 The ensuing enhancement of circulation half-life (T_1/2) of the stealth liposomes enables greater drug exposure, reduced toxicity, and less frequent administration. Occasionally, the clinical requirement itself demands extended functional life of therapy, as in the case of liposome-based oxygen carriers simulating red blood cells.^10,11

Among the synthetic materials, polyethylene glycol (PEG)–phospholipids are the most commonly employed materials for creating stealth liposomes.¹² Doxil^®, a drug for ovarian cancer, multiple myeloma, AIDS-related Kaposi’s sarcoma, and many other cancers, is a PEGylated liposome preparation of Adriamycin (also known as doxorubicin).¹³ PEG affords a nonionic character, high solubility in both aqueous and organic media, biocompatibility, low immunogenicity, and good excretion kinetics.^14–18 PEGylated liposomes demonstrate circulation T_1/2s of 15–24 h in rodents and 45 h in humans.¹⁹ Although liposomes containing PEG–phospholipids have successfully reached the clinic, significant problems have been attributed to phospholipid anchors in these amphiphiles.²⁰ In fact, both phospholipid and PEG components of PEG–phospholipids have been identified as causes of nonspecific immune reactions in vivo.¹² These reactions are ascribed to the presence of a negatively charged phosphoryl group in clinically used PEG–phospholipids,^21–23 variability in size, and the nature of acyl anchor.²⁴ PEG itself is not completely innocuous in biological systems as it has been found to induce complement-mediated hypersensitivity reactions.^25,26 Its administration results in the production of anti-PEG IgM antibodies against the interface between hydrophilic PEG and hydrophobic lipid.²⁷ Specifically, the phosphate oxygen moiety of a phospholipid–methoxy(polyethylene glycol) conjugate plays a critical role in immune-related consequences of PEGylated liposomes.²¹ Anti-PEG antibodies play a major role in the clearance of intravenously administered PEGylated liposome products, especially upon repeated administration.²⁸ Moreover, PEGs with molecular weight less than 400 Da may undergo alcohol dehydrogenase-catalyzed oxidation, resulting in toxic diacid and hydroxy acid metabolites.²⁹ On the contrary, very large PEGs with sizes exceeding the cut-off of renal clearance (30–50 kDa) have limited biodegradability, resulting in accumulation after dosing.^30,31

Although the amphiphilic character is the basis of the utility of PEG-phospholipids in liposome-surface modification, PEG molecules themselves contain both hydrophilic as well as hydrophobic domains. The hydrophobic domain of PEG interacts with the underlying phospholipid bilayer and prevents the hydration of membrane phospholipid headgroups, resulting in poor drug loading and destabilization of liposomes.^32,33 PEGylation also impacts the physical stability of liposomes during cryprotectant-free lyophilization cycles.^34,35

In light of the above-mentioned revelations, we contemplate that despite overall safe and effective performance of PEG–liposomes, there are a few physicochemical and physiological consequences of liposome PEGylation, and an opportunity exists for the development of better hydrophilic lipopolymers. In general, interest is growing in the development of nonphospholipids and PEG–nonphospholipids amphiphiles.^36–41 Recently, we reported PEG₂₀₀₀ conjugated with N¹-(2-aminoethyl)-N⁴-hexadecyl-2-tetradecylsuccinamide (HDAS–PEG) as a replacement of PEG–phospholipids; HDAS–PEG modification demonstrated significantly reduced liposome-induced complement activation as compared with the more commonly used DSPE-PEG.⁴¹ In this contribution, we present the evaluation of a novel non-PEG–nonphospholipid conjugate for enhanced circulation persistence of liposomes. The stealth behavior of this conjugate is imparted by a superhydrophilic polymer (SHP), poly [N-(carboxymethyl)-2-(isobutyryloxy)-N,N-dimethylethanammonium], whereas HDAS was retained as a lipid anchor from our previous works.^41,42 Results demonstrate that liposomes modified with N¹-(2-aminoethyl)-N⁴-hexadecyl-2-tetradecylsuccinamide conjugated with SHP (HDAS–SHP) circulate in blood for a prolonged time period, with significantly reduced activation of complement system. Furthermore, the modification does not adversely affect pH-gradient-mediated active loading of Adriamycin inside the liposomes.

EXPERIMENTAL

All the chemicals were purchased from Sigma–Aldrich (St. Louis, Missouri) and/or various suppliers represented by VWR Scientific (West Chester, Pennsylvania), and, unless otherwise mentioned, they were used without further purification. 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA) was acquired from Santa Cruz Biotechnology (Dallas, Texas). For liposome preparation, phospholipids were purchased from Lipoid (Ludwigshafen, Germany), Avanti Polar Lipids (Alabaster, Alaska), or NOF Corporation (Tokyo, Japan). High-purity cholesterol (CHO) was obtained from Calbiochem (Gibbstown, New Jersey). ¹H nuclear magnetic resonance (NMR) spectra were recorded at 300 and 75 MHz on Mercury-VX 300 (Varian Inc., Palo Alto, California).

Synthesis of HDAS–SHP

Scheme 1 shows the synthetic procedure for HDAS–SHP. The synthesis of acyl chain precursor, 2-hexadecylcarbamoylmethyl hexadecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (1, HDAS–NHS) has been previously reported.^41,42 N-hydroxysuccinimide 2-bromopropanoate (4) and 2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanammonium (5) were synthesized (Supplemental Data) according to the published procedures.⁴³ The precursor polymer was synthesized by atom transfer radical polymerization (ATRP).

Scheme 1.

Synthetic scheme for HDAS–SHP (7). Reagents and conditions: (a) N-Boc–ethylenediamine, dichloromethane, room temperature, 24 h. (b) Trifluoroacetate, room temperature, 8 h. (c) ATRP; Cu(I)Br/HMTETA, DMF, 60°C, 24 h. (d) 3, dimethylsulfoxide, 45°C, 72 h. (e) Trifluoroacetate, room temperature, 8 h.

Tert-Butyl (2-(2-(2-(Hexadecylamino)-2-Oxoethyl)Hexadecanamido)Ethyl)Carbamate (2)

Compound 1 (HDAS–NHS, 3.0 g, 4.72 mmol) and N-Boc-ethylenediamine (0.91 g, 5.67 mmol) were dissolved in anhydrous dichloromethane (60 mL) and the reaction mixture was vigorously stirred for 24 h at room temperature. White solid precipitate appeared upon completion of the reaction. The solvent was removed by a rotavapor and the product was purified by washing with cold methanol (3 × 20 mL). The purified product (2) was dried under vacuum, producing white powder (3.10 g, yield 96%). ¹H NMR (300 MHz, CDCl₃) δ (ppm): 6.39 (br, 1H, NH), 5.92 (br, 1H, NH), 5.07 (br, 1H, NH), 3.49–3.09 (m, 6H), 2.87–2.31 (m, 3H), 1.80–1.40 (br, 6H), 1.43 [s, 9H, O-C(CH₃)₃], 1.35–1.15 (br, 48H, CH₂), 0.87 (t, 6H, CH₃). ESI–MS calculated for C₄₁H₈₁N₃O 679.62, found 680.6 [M+H]⁺.

N¹-(2-Aminoethyl)-N⁴-Hexadecyl-2-Tetradecylsuccinamide (3)

The terminal NH₂ functionality of 3 was obtained by simply deprotecting 2 using trifluoroacetic acid (TFA). Briefly, compound 2 (3.0 g, 4.41 mmol) was dissolved in a mixture of dichloromethane and TFA (1:4 ratio, 50 mL) and the reaction was allowed to continue for 8 h. After removing the solvent with a rotavapor under reduced pressure, the product was dissolved in methanol and kept at 0°C for precipitation. The precipitate was filtered and dried to obtain the white powder (2.7 g, 88%). ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.80 (br, 2H, NH₂) 6.39 (br, 1H, NH), 5.92 (br, 1H, NH), 5.07 (br, 1H, NH), 3.49–3.09 (m, 6H), 2.87–2.31 (m, 3H), 1.80–1.40 (br, 6H), 1.35–1.15 (br, 48H, CH2), 0.87 (t, 6H, CH₃). ESI–MS calculated for C₃₆H₇₃N₃O₂ 579.57, found 580.6 [M+H]⁺.

NHS–Polymer (6)

The ATRP of acrylic monomer 5 and NHS-activated macroinitiator 4 was carried out in nitrogen atmosphere using a Schlenk tube. Compound 4 (0.25 g, 1 mmol), acrylic monomer 5 (3.45 g, 12.66 mmol), and HMTETA (0.272 mL) were dissolved in degassed dimethylformamide (DMF). The solution was further degassed by three cycles of positive nitrogen flow and vacuum. After degassing, Cu(I)Br (142 mg) was added to the reaction mixture against the nitrogen flow. The reaction was allowed to continue at 60°C for 24 h under nitrogen flow. The polymeric product was precipitated into acetone (2 × 200 mL) to remove the catalyst and residual monomers. The resulting precipitate was dried under vacuum to obtain compound 6 (3.0 g, 51%). ¹H NMR (300 MHz, D₂O) δ (ppm): 4.54–4.18 (br, 92H, -OCH₂CH₂N(CH₃)₂CH₂COO-), 4.13–3.80 (br, 46H, -OCH₂CH₂N(CH₃)₂CH₂COO-), 3.34 (br s, 138H, -OCH₂CH₂N(CH₃)₂CH₂COO-), 2.84 (t, 4H, -COCH₂CH₂CO- from NHS, initiator), 2.10–1.1.60 (br, 47H, -OOCCH(CH₃)CH₂C(CH₃)OCH₂-), 1.45 (br s, 208H, -OC(CH₃)₃). 1.29–0.80 (br, 72H, -OOCCH(CH₃)CH₂C(CH₃)-(Br)/OCH₂-).

N¹-(2-Aminoethyl)-N⁴-Hexadecyl-2-Tetradecylsuccinamide Conjugated with SHP (7)

N¹-(2-aminoethyl)-N⁴-hexadecyl-2-tetradecylsuccinamide 3 (0.173 g, 0.25 mmol) and polymer 6 (0.5 g) were dissolved in 20 mL of dimethyl sulfoxide (DMSO) at 45°C. To the reaction mixture, 0.10 mL of trimethylamine was added and the mixture was stirred vigorously for 72 h. The product was purified by dialysis using cellulose tubing (MW cut-off 1000 Da; Sigma–Aldrich) against DMSO and water for 24 and 48 h, respectively. Finally, the dry protected HDAS–SHP was obtained (0.24 g, 63%) by removing water through lyophilization (Triad Lyophilizer; Labconco, Kansas City, Missouri). HDAS–SHP (7) was obtained by simply deprotecting the precursor polymer–lipid conjugate. Briefly, the protected precursor was dissolved in 6 mL of TFA and stirred for 8 h at room temperature. After removing TFA with a rotavapor under reduced pressure, the product was purified by dialysis using cellulose tubing (MW cut-off 1000 Da) against methanol and water for 24 and 48 h, respectively. The aqueous solution of the product was filtered and dried by lyophilization, producing 0.30 g (yield 70.0%) of fluffy powder. The formation of HDAS–SHP was confirmed by NMR (Fig. 1a). ¹H NMR (F₃COOD) δ (ppm): 5.08–3.83 (br, 138H, -OCH₂CH₂N(CH₃)₂CH₂COO-), 3.55 (br s, 138H, -OCH₂CH₂N(CH₃)₂CH₂COO-), 3.40–2.78 (m, 9H), 2.50–1.90 (br, 47H, -OOCCH(CH₃)CH₂C(CH₃)OCH₂-), 1.80–1.50 (br, 6H), 1.56–0.96 (br, 122H, -OOCCH(CH₃)CH₂C(CH₃)-(Br)/OCH₂-) and CH₂ from the acyl chain), 0.89 (t, 6H, CH₃).

Figure 1.

(a) ¹H NMR spectrum of NHS–polymer 6 (upper panel) and HDAS–SHP 7 (lower panel) in D₂O and F₃COOD, respectively. (b) DSC thermogram of HDAS–SHP, showing values of peak maxima for glass transition temperature (T_g) and melting temperature (T_m).

Characterization of HDAS–SHP

In addition to the usual NMR and mass spectroscopic measurements described above, we submitted the samples of purified HDAS–SHP for gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). GPC was performed by FAI Materials Testing Laboratory, Inc. (Marietta, Georgia), whereas DSC services were provided by Photometrics, Inc. (Huntington Beach, California). From GPC measurements, the MW of the polymer was determined against PEG standards using HP1090 Series II Liquid Chromatograph combined with a Waters UltraHydrogel linear column and Waters 2410 Refractive Index Detector. Phosphate buffer (PBS) was used as an eluent with a flow rate of 1 mL/min. The DSC thermograms were obtained on a 2920 Modulated DSC machine (TA Instruments, New Castle, Delaware) with a refrigerated cooling system. The calibration of the equipment for temperature and enthalpy was performed using indium. Briefly, samples (5–10 mg) were placed in aluminum pans and nitrogen gas was purged at 40 mL/min. An empty aluminum pan was used as a reference. Samples were heated from −20°C to 180°C at a rate of 10°C/min. The data were analyzed using the “TA Universal Analysis” software.

Preparation of Liposomes Modified with HDAS–SHP

Liposome Preparation

Lipid hydration followed by extrusion was employed for the preparation of liposomes. Briefly, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (0.247 g; DPPC), 1,2-ditetradecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (0.051 g; DMPG), CHO (0.129 g), and vitamin E (64.5 μL of 10 mg/mL solution) in 45:10:44.8:0.2 mol % were dissolved in a mixture of 20 mL chloroform–methanol (2:1), filtered through 0.2-μm nylon filter, and transferred into a round bottom flask. The solvent mixture was evaporated at 45°C on an R-210 Rotavapor (Buchi, Flawil, Switzerland) to form a thin film of lipid mixture. Any trace of organic solvents was removed by keeping the film under vacuum for additional 12 h. The lipid film was rehydrated with water, and the suspension was lyophilized overnight (Triad Lyophilizer; Labconco) to create proliposomes. The dried mixture was again hydrated with 15 mL of 100 mM aqueous solution of glutathione (pH 6.8–7.0, adjusted with dilute NaOH). The lipid suspension was sequentially extruded (Lipex Biomembranes Inc., Vancouver, Canada) using polycarbonate membranes of pore sizes 1, 0.6, 0.4, and 0.2 μm. The extrusion was performed at 55°C and repeated at least five times for each pore size. The final preparation of liposomes was divided into two equal halves. One-half of the preparation was subjected to HDAS–SHP modification and the other half was stored at 4°C as plain liposomes.

Insertion of HDAS–SHP in Preformed Liposomes

Insertion of HDAS–SHP into the external layer of liposomes was performed essentially by the method reported previously.^41,44 Briefly, 0.04 mmol (equivalent to ~8% mmol of the total phospholipids in the liposomes) of HDAS–SHP was dissolved in 50 mL of water and filtered through 0.2 μm. The solution was added to the diluted (five times) suspension of preformed liposome at a rate of 50 μL/min using a programmable syringe pump (Chemyx Inc., Satfford, Texas) at room temperature. During the addition of HDAS–SHP, the suspension was stirred moderately in an inert (N₂) atmosphere. The preparation was concentrated by centrifugation at 184,500g for 45 min at 4°C (Optima L-100 XP Ultracentrifuge; Beckman Coulter, Fullerton, California). The supernatant was removed, the pellet was resuspended in PBS, and the centrifugation cycle was repeated two more times to completely eliminate extravesicular glutathione and unincorporated HDAS–SHP. The second half of liposome preparation (plain liposomes) was identically handled except that the HDAS–SHP solution was replaced by sterile water in these liposomes. The final pellets of HDAS–SHP liposomes and plain liposomes were resuspended in PBS (pH 7.4) containing 300 mM sucrose and stored at 4°C until further use.

Physicochemical Characterization of Liposomes

The phospholipid concentration in the liposome preparations was determined by calorimetry⁴⁵ using Perkin-Elmer Lambda 4B UV/Vis spectrophotometer (Waltham, Massachusetts). Particle size, size distribution, and zeta potential of the liposomes were studied with dynamic light scattering (DLS) using a Zeta PLUS analyzer (Brookhaven Instruments Corporation, Holtsville, New York) equipped with 532 nm laser. The Zeta Potential and DLS were measured in aqueous phases of pH 6.8 and 2.0. The desired pH of the solutions was achieved using diluted hydrochloric acid. To visualize the liposomes, we performed electron microscopy at the Oklahoma Medical Research Foundation’s Imaging Core Facility (Oklahoma City, Oklahoma). The electron micrographs were obtained using a Hitachi H-7600 Electron Microscope at 30,000× operating at 80 kV. Briefly, a dilute suspension of liposomes was placed on a formvar-coated copper grid (400 meshes) and allowed to stand for 3 min in air. The excess sample was removed, followed by negative staining with 1% aqueous uranyl acetate solution. The stained grid was allowed to air dry for 5 min before the microscopy.

Radiolabeling of Liposomes

The presence of encapsulated glutathione enables the labeling of liposomes with ^99mTc-labeled hexamethylpropyleneamine oxime (^99mTc-HMPAO) using a procedure described previously.⁴⁶ Freshly prepared ^99mTc-HMPAO was obtained from OUHSC-Nuclear Pharmacy (Oklahoma City, Oklahoma). ^99mTc-HMPAO (0.25 mL, ~370 MBq) was added to 1.50 mL of liposomes and incubated for 30 min at room temperature, with occasional swirling. The labeled liposomes were passed through a PD-10 column (GE Healthcare Life Sciences, Pittsburg, Pennsylvania) to separate any radioactivity not associated with the liposomes. The labeling efficiency of column purified ^99mTc-liposomes was determined by performing paper chromatography in saline and found to be more than 95% for all preparations.

Active Loading of Adriamycin

We used ammonium sulfate pH-gradient DSPC/CHO (molar ratio 55:45) liposomes obtained from FormuMax Scientific, Inc. (Palo Alto, California) to investigate the effect of surface modification with HDAS–SHP, DSPE–PEG₂₀₀₀, and HDAS–PEG₂₀₀₀⁴¹ on Adriamycin loading. We chose this as a test system because of its proven success^47,48 and PEGylated liposome formulations of Adriamycin prepared using this technique are currently in clinical use.¹³ First, we exchanged the external medium of the liposomes with 250 mM (NH₄)₂SO₄ by using a PD 10 column. The resultant liposome preparation, with in/out (NH₄)₂SO₄, was divided into four parts. The first three parts of the liposomes were postinserted with HDAS–SHP, HDAS–PEG₂₀₀₀, and DSPE-PEG₂₀₀₀ lipids solution in 250 mM (NH₄)₂SO₄, respectively, whereas the fourth part was kept as a plain control. The postinsertion of these amphiphiles was performed according to the procedure described above. For Adriamycin loading, the external (NH₄)₂SO₄ of surface-modified and plain liposomes was removed by buffer exchange using isotonic HEPES-buffered saline (10 mM of HEPES and 370 mM of NaCl, pH 7.4). The resultant liposomes, with in (NH₄)₂SO₄/out HEPES and final phospholipid concentration of about 5 mM, were used for active loading of Adriamycin. Briefly, a 1-mM solution of Adriamycin HCl in HEPES buffer was added to the liposomes. Three lipid-to-drug ratios (2.0, 5.0, and 10.0 mmol/mmol ratios) were tested, while keeping the total lipid concentration constant (1.62 mM). The loading process was carried out by incubating the mixture at 37°C or 55°C with moderate swirling for about 20 h. The loading experiments were triplicated for each liposome preparation.

Determination of Encapsulation Efficiency

The separation of Adriamycin-loaded liposomes from free Adriamycin was performed by size exclusion chromatography using a PD 10 column. Briefly, 0.4 mL of the incubated mixture was passed through the column using HEPES buffer. The free Adriamycin and liposome-encapsulated Adriamycin appeared in clearly visualized separate bands. The liposomes were carefully collected and diluted to 1.5 mL with HEPES buffer. For determination of encapsulated Adriamycin, liposomes were lysed with Triton X-100 (0.5%, v/v)⁴⁹ and assayed for Adriamycin by measuring the optical density at 480 nm and comparing it with predetermined molar absorptivity (10,668 M⁻¹/cm) using a Perkin-Elmer Lambda 4B UV/Vis spectrophotometer.

The retention of Adriamycin in different types of liposomes was determined by storing them for 1 month at 4°C and 25°C and estimating the leaked Adriamycin by size-exclusion chromatography. Again, PD 10 column chromatography was employed to separate leaked drug from intact liposomes. Percent retention was calculated as a ratio of liposome-associated Adriamycin to total Adriamycin in the original liposomes.

Animal Studies

The animal experiments were performed according to the National Institutes of Health Animal Use and Care Guidelines and were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Science Center.

Imaging and Tissue Distribution Studies

Sprague–Dawley rats (male, 225–250 g) were divided in two groups of five animals each. The first group received ^99mTc–HDAS–SHP liposomes, whereas the second group received ^99mTc-Plain liposomes. All injections (18.5 MBq, 0.5 mL and ~6.0 mg phospholipid dose) were performed in the tail vein of rats under 2% isoflurane anesthesia (Butler Schein Animal Health, Dublin, Ohio) in a stream of oxygen. After radioactivity administration, anesthetized rats were imaged by a NanoSPECT machine (Bioscan Inc., Washington, District of Columbia) at various times before euthanasia with an overdose of Euthasol (Virbac Corporation, Fort Worth, Texas) at 24 h postinjection. Various organs were excised, washed with saline, weighed, and appropriate tissue samples were counted in a gamma counter (PerkinElmer Life and Analytical Sciences, Boston, Massachusetts). The femur with bone marrow was taken as representative of bone tissue. Total blood volume, bone, and muscle mass were estimated at 5.4%, 10%, and 40% of body weight, respectively. A diluted sample of injected liposomes served as a standard for comparison.

Complement Activation

We examined activation of complement pathway by determining C3a (classical marker) and SC5b (S protein-bound terminal complex) using MicroVue complement enzyme-immunoassay kits (Quidel, Santa Clara, California). Blood was withdrawn from rats after 1 h of HDAS–SHP or plain liposomes injection. Same amount of phospholipid (6 mg) was injected in the two groups. Samples were centrifuged and plasma was assayed following manufacturer’s instructions.

Data Analysis

The data were statistically analyzed by the two-way analysis of variance using GraphPad Prism 6.0 software for Windows (La Jolla, California). For multiple comparisons, Bonferroni’s post-hoc test was applied. All average values were given ±standard error of mean. The acceptable probability for significance was p < 0.05. To obtain profiles of liposome distribution in blood, the gamma camera images were analyzed by drawing regions of interest (ROI) around heart voxels. The radioactive counts were normalized by the counts associated with the whole body and plotted with respect to time. All calculations pertaining to ^99mTc radioactivity involved correction for decay of ^99mTc (T_1/2 6.0 h).

RESULTS AND DISCUSSION

We report the synthesis of HDAS–SHP (7) as a novel non-PEG and nonphospholipid alternative to phospholipid-linked PEG polymers for the surface modification of liposomes. Unlike phospholipids, the HDAS component of this new superhydrophilic lipopolymer contains no phosphoryl moiety that has been attributed with hypersensitivity reactions observed with the usage of liposome products.^22,23 The resultant surface-modified liposomes were tested in a rat model to demonstrate the utility of HDAS–SHP in reducing liposome-mediated complement activation and enhancing the circulation persistence of the liposomes.

The SHP portion of 7 was synthesized as an NHS ester (6) by a reaction between ATRP macroinitiator N-hydroxysuccinimide 2-bromopropanoate and 2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanammonium. The degree of polymerization (DP) of 6 was determined to be approximately 23 by comparing the area of ¹H NMR peaks (Fig. 1a) at 3.38 ppm for –OCH₂CH₂N(CH₃)₂CH₂COO and 2.84 ppm for –COCH₂CH₂CO–. Accordingly, the molecular weight of 6 was calculated to be 5,870 Da. GPC measured the number–average molecular weight of 6 as 5,620 Da with polydispersity index of 1.22. The SHP polymer 6 was conjugated to an amine-ended N¹-(2-aminoethyl)-N⁴-hexadecyl-2-tetradecylsuccinamide to create an amphiphilic polymer HDAS–SHP (7; Scheme 1). The ratio of HDAS-to-SHP was determined to be approximately 1/23 that was in good agreement with the DP of NHS–polymer determined via NMR. According to the ¹H NMR (Fig. 1a), the MW for HDAS–SHP was calculated to be 5042 Da. HDAS–SHP demonstrated a clear endothermic transition peak in the DSC thermogram at 44°C that could be assigned to a glass transition temperature (T_g), indicating its amorphous morphology (Fig. 1b). The T_g was followed by a melting temperature (T_m) at 149.5°C.

Structurally, HDAS–SHP possessed a sharp disparity in polarity because of the two acyl hydrophobic chains on one end and a long polymeric backbone composed of zwitterionic side chains on the other end. The zwitterionic nature is afforded by multiple repeats of quaternary ammonium and carboxylate groups (7; Scheme 1), which also provides a highly hydrophilic character to the polymer. Quaternary ammonium ion possesses permanent positive charge in a wide range of pH values,^50,51 whereas the carboxylate ion shows negative charge or no charge depending on the pH.⁵² This is distinct from PEG–phospholipids that have no permanent charge and possess amphiphilic PEG chains with significant hydrophobicity. Because of the presence of hydrophobic domains within the PEG chain, there exists a tendency for the lipid bilayers to become destabilized. Second, in contrast to the hydration of the PEG chains via hydrogen bonding, SHP is strongly hydrated by water molecules through electrostatic interactions. The resultant enhancement in hydrophilicity is expected to reduce the interactions of SHP with opsonizing biomolecules or cells. This type of SHP exhibits ultralow fouling and very strong hydration properties in biological milieu.^53,54 Recently, Cao et al.⁴³ reported a SHP conjugated with DSPE for the stabilization of liposomes.

We used HDAS–SHP to modify the surface of preformed liposomes. The negatively stained electron micrographs revealed the spherical nanostructure formations with average diameter below 200 nm, which was expected after extrusion through a 200-nm polycarbonate filter (Figs. 2a and 2b). The microscope-based mean diameter measurements were in good agreement with the particle size determination made using DLS. The narrow polydispersity of the preparations suggests that liposomes exhibited a homogeneous size distribution. The electron micrographs of HDAS–SHP-modified liposomes also revealed a white circle surrounding the spherical body of the liposomes, which is indicative of the presence of the polymeric layer on the surface (Fig. 2b).

Figure 2.

Electron micrographs of (a) plain and (b) HDAS–SHP liposomes. DLS for particle size distribution of (c) plain and (d) HDAS–SHP liposomes.

The physicochemical characteristics of the liposome preparations are listed in Table 1. A relatively high-negative Zeta potential of plain liposomes was reduced by approximately 21 mV after modification with HDAS–SHP, suggesting that HDAS–SHP occupied the liposome surface. In addition, HDAS–SHP-modified liposomes showed a drastic change (35 mV; Table 1) in the Zeta potential when pH of the medium was changed from 6.8 to 2.0. In contrast, the plain liposomes retained the negative potential independent of the pH of the medium. The pH-dependent change in Zeta potential is a characteristic phenomenon of the surface-modified particles carrying zwitterionic charge. In the case of HDAS–SHP, the carboxylate groups on the polymer exist as protonated and deprotonated species at pH 2 and 6.8, respectively; however, the positive charge from the quaternary ammonium ions remains revealed at both pH values.

Table 1.

Physicochemical Characteristics of Plain and HDAS-SHP-Modified Liposomes with Core Composition of DPPC/DMPG/CHO/Vit-E (45:10:44.8:0.2 mol %)

	Diametera (nm, Polydispersity)		Zeta Potential (ζ mV)		Δζ(mV)	Phospholipid (mg/mL)	99mTc-Labeling Efficiency (%) Before SECb
	pH		pH
	6.8	2	6.8	2
Plain liposomes	154 (0.07)	172 (0.07)	−32.1	−20.2	11.9	13.6	~80
HDAS-SHP liposomes	167 (0.07)	158 (0.08)	11	24.5	34.5	11	~80

^aDynamic light scattering.

^bSize-exclusion chromatography.

The primary goal of modifying the liposomal surface was to prolong circulation persistence of liposomes and reduce their MPS uptake, secondary to opsonization with complement proteins. HDAS–SHP was postinserted into the outer lipid layer of liposomes, and liposomes were labeled with a gamma ray-emitting ^99mTc radionuclide for in vivo monitoring over a period of 24 h after i.v. administration. The dynamic images (Fig. 3a) taken immediately after injection revealed the difference in blood pool (the image signal emanating from heart) between plain liposomes and HDAS–SHP liposomes within 5 min of injection. By 12 h postinjection (and more so by 24 h), the majority of plain liposomes were cleared from circulation, primarily accumulating in liver and spleen. On the contrary, a significant amount of injected HDAS–SHP liposomes persisted in circulation. The early (45 min) accumulation of ^99mTc radioactivity in the urinary bladder of rats injected with plain liposomes indicates the release of ^99mTc radioactivity after metabolic processing of liposomes in the MPS.

Figure 3.

Demonstration of enhanced circulation persistence of HDAS–SHP-modified liposomes in comparison to the circulation persistence of the plain liposomes. (a) Representative scintigraphic images of rats injected with ^99mTc-labeled liposomes have been shown. (b) The blood pool profile obtained by quantitating the radioactivity signal emanating from the heart region.

ROI analyses of images revealed that compared with 5.3% (±0.8, SEM) of the plain liposomes, approximately 28.9% (±3.6, SEM) of administered HDAS–SHP liposomes were present in the blood after 24 h postinjection (Fig. 3b). We performed analysis of data by employing a two-compartmental model, and found that α (fast phase) and β (slow phase) values for the HDAS–SHP liposomes were 5.93 and 0.014 h⁻¹, respectively; the α and β for plain liposomes were calculated to be 5.54 and 0.064 h⁻¹, respectively. This suggests that the initial clearance of both the liposome preparations was similar, but the clearance of HDAS–SHP liposomes was considerably reduced during the prolonged slow phase of elimination from blood (0.5–24 h). Similar ROI analysis of images obtained in our previously reported work⁴¹ with DSPE–PEG-modified liposomes showed 18.0% (±3.42, SEM) of injected DSPE–PEG liposomes in blood after 24 h, with α and β values of 6.4 and 0.042 h⁻¹, respectively. Additional study is necessary to directly compare the pharmacokinetics of HDAS–SHP liposomes and DSPE—PEG liposomes under identical conditions.

The biodistribution of liposomes at necropsy is shown in Figure 4. Besides blood pool, the major organs of radioactivity accumulation were spleen, liver, bone, and kidney; other organs accumulated negligible amount of radioactivity. Significantly more HDAS–SHP liposomes (~10 times) were measured in the blood as compared with the plain liposomes. Similar comparison of data from our previous report revealed that DSPE–PEG-modified liposomes were approximately seven times more in the blood as compared with the plain liposome after 24 h of injection.⁴¹ Although the levels of plain and HDAS–SHP liposomes accumulation in the MPS (liver and spleen) appear similar, clearance of the MPS-associated plain liposomes from the body had occurred by 24 h when the necropsy was performed. The biodistribution of HDAS–PEG liposomes in various organs was similar to that of DSPE–PEG liposomes (reported elsewhere)⁴¹; however, the circulation-bound liposomes were significantly more in the former than in the latter. Overall, results indicate that HDAS–SHP conferred stealth characteristics to the liposomes and increased their apparent circulation half-life. Both the images and the biodistribution data were consistent with the previously published reports on plain and PEG liposomes.^44,55–57

Figure 4.

Biodistribution of HDAS–SHP-modified liposomes and plain liposomes in rats at 24 h (n = 5 for each group). Percent injected dose per gram of tissue (±SEM) is shown (*p < 0.05 vs. plain liposomes).

The difference between the circulation persistence of HDAS–SHP liposomes and plain liposomes could be attributed to reduced opsonization of the former by serum complement proteins. Complement proteins have been previously identified as being responsible for liposome clearance from circulation.^58–60 We found that HDAS–SHP has a lower tendency of activating complement in vivo than DSPE–PEG and HDAS–PEG. The levels of C3a (classical marker) and SC5b-9 (S protein-bound terminal complex) in serum is shown in Figure 5. Results clearly suggest that postinsertion of HDAS–SHP significantly subdued the liposome-induced complement activation. Whether the affect on complement system alone is responsible for enhanced circulation persistence of HDAS–SHP liposomes remains an open question because other factors such as resistance of HDAS–SHP to metabolic processing or reduced loss of HDAS–SHP from the liposome surface cannot be ruled out as additional reasons.

Figure 5.

Complement activation by HDAS–SHP-modified liposomes and plain liposomes. Plasma was collected from rats after 1 h of liposome injection. Complement protein C3a plus and Sc5B complex were measured by ELISA. Results are an average of three replicates and the error bar represent SEM (*p < 0.05).

High encapsulation efficiency (EE) at a low lipid-to-drug ratio is desirable for liposome-based drug delivery, because the in vivo toxicity of liposomes diminishes with a decrease in the lipid-to-drug ratio.⁶¹ We investigated whether the external zwitterionic SHP would affect the transmembrane ammonium sulfate gradient-driven active loading of Adriamycin in HDAS–SHP-modified liposomes. For comparison, plain liposomes and liposomes modified with HDAS–PEG₂₀₀₀, or DSPE–PEG₂₀₀₀ were also studied. The characteristics of these liposomes are provided in Table S1 (Supplemental Data). As shown in Figure 6, the EE was dependent on lipid-to-drug ratio, especially at the more discriminatory temperature of 37°C. In general, all the liposomes showed low loading performance with the lipid-to-drug ratio of 2 at 37°C. HDAS–PEG and DSPE–PEG liposomes showed only 43% and 23% EE₃₇, respectively, whereas both plain and HDAS–SHP liposomes showed approximately 70% EE₃₇. By increasing the lipid-to-drug ratio to 10, the EE₃₇ for all the preparations was found to increase, but the mean EE₃₇ in HDAS–PEG and DSPE–PEG liposomes remained lower than that in plain and HDAS–SHP liposomes. However, at 55°C and with higher lipid-to-drug ratios, all the liposomes demonstrated high EE₅₇ (Fig. 6; Supplemental Table S1). For the maximum encapsulation in DSPE–PEG liposomes, the lipid-to-drug ratio of 5–10 (M ratio) was required. In comparison, HDAS–SHP could reach equivalent encapsulation at a lipid-to-drug ratio of only 2 (M ratio). Therefore, in addition to the stealthiness, HDAS–SHP also provides high encapsulation at a lower lipid concentration than what could be achieved by using the traditional DSPE–PEG. Poor drug encapsulation in PEGylated liposomes has also been reported in literature; incorporation of 6 mol % of DSPE–PEG₂₀₀₀ decreased the EE to 63%.³³ We hypothesize that the external PEG chains create a hindrance to the transmembrane localization of Adriamycin, and this hindrance is mitigated by loading the drug at a temperature close to the melting temperature of the lipid. At 55°C, the lipid constituents afforded maximum fluidity to the liposome membrane. In contrast, significantly more Adriamycin could be loaded in HDAS–SHP liposomes even at a lower temperature (37°C).

Figure 6.

Encapsulation efficiency(%) of Adriamycin in plain liposomes and liposomes modified with HDAS–SHP, HDAS–PEG, and DSPE–PEG at (a) 37°C and (b) 55°C. The core composition of the liposomes consisted of DPPC and CHO and the drug loading was assisted by transmembrane pH gradient. Each experiment was performed in triplicate and the error bars represent SEM (*p < 0.05 vs. plain and ^#p < 0.05 vs. HDAS–SHP).

These encapsulation experiments reveal that compared with DSPE–PEG liposomes, loading of Adriamycin in HDAS–SHP liposomes was relatively less sensitive to the processing temperature, allowing for substantial loading at lower temperatures. This phenomenon might be an attractive proposition for the encapsulation of temperature-sensitive drugs. To this extent, we speculate that replacement of PEG phospholipid with HDAS–SHP would not only improve the therapeutic index of Adriamycin liposomes, but would also be an advantage at the formulation bench. Furthermore, HDAS–SHP liposomes retained approximately 95% of Adriamycin after storage for 1 month at both 4°C and room temperature (Fig. 7). The liposomes remained stable in suspension without any visible settling. The size and polydispersity of the loaded liposomes were comparable to those of the original liposomes (Table S1, Supplemental Data).

Figure 7.

Retention of Adriamycin in plain, HDAS–SHP-, HDAS–PEG-, and DSPE–PEG-modified (DPPC/CHO) liposomes after storage for 1 month at 4°C or 25°C. Each experiment was repeated ≥4 times and error bars represent SEM.

CONCLUSIONS

We report a novel HDAS–SHP for imparting stealth properties to liposomes. Liposomes modified with HDAS–SHP demonstrated improved encapsulation of Adriamycin by the active loading method, provided enhanced circulation persistence after administration, and were found to be less susceptible to induce complement activation. Given the increasing use of PEG in pharmaceuticals, cosmetics, and foods products, anti-PEG antibodies have been detected even in healthy individuals without any prior exposure to PEG in dosage forms.^62,63 These anti-PEG IgMs play a role in clearance and immunereactivity of PEGylated liposomes. Non-PEG nonphospholipid HDAS–SHP lipopolymer offers an alternative means of imparting stealth characteristics to the liposomes and lipid-based nanoparticles.

Supplementary Material

This article contains supplementary material available from the authors upon request or via the Internet at http://wileylibrary.com.