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Amphiphilic poly- N-vinylpyrrolidones
Abstract
Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4′-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low μmolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1500 and 8000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.
... Triggered-delivery, improved drug internalization, and higher anticancer effects have been observed with these PEG alternatives [126]. Few other alternative polymers, with enhanced physiological properties, are acrylic polymers poly(N-(2-hydroxyethyl) acrylamide) (polyHEAA) and phosphatidyl polyglycerols [127][128][129][130][131]. ...
... Apart from drugs, small molecules and nucleic acids can also be carried to the target area, gaining molecular level approach [145][146][147][148]. Another novel but fascinating development, to reach mitochondria through active targeting surface by means of molecules and peptides, such as biguanide and guanidine, has widened the application potential of liposomes [66,149]. Access to the cell can also be achieved by binding penetratin, phosphoenolpyruvate-1 (PEP-1), and polyarginine to liposomes [130,150]. Very specific ligand- targeted liposomes through previously mentioned specific ligands, such as mAb or peptides, attaching directly to liposomes or to PEG are also attainable [151]. ...
... PVP is a wellknown water-soluble and biocompatible polymer. [21][22][23][24] The PVP amide group easily forms hydrogen bonds and as a result, PVP has a high binding affinity for several small and large molecules. 25,26 In our previous studies, we have shown that PVP amphiphilic derivatives form spherical nano-scaled micelle-like assemblies (30-200 nm) at considerably low concentrations in aqueous media, which can effectively incorporate various lowmolecular and macromolecular drugs. ...
... Increase in the concentration of mercaptoacetic acid was found to decrease the molecular weight of the resulting polymer. 24 In the second step, coupling of a long-chain hydrophobic n-octadecyl moiety with the carboxy-terminal functional group of the semitelechelic PVP macromolecules was performed via N,N'-Dicyclohexylcarbodiimide (DCC) coupling. As a result, the amphiphilic polymers prepared for the current investigation contained water-soluble PVP blocks and one, nonpolymeric, hydrophobic octadecyl end group (Figure 1). ...
... This fact is associated with their ease of preparation, low toxicity, and the ability to control the hydrophilicity (hydrophobicity) of the synthesized chains. Considerable prospects due to high biocompatibility and low toxicity [16,17] are associated with the use of carriers based on amphiphilic oligomers of N-vinyl-2-pyrrolidone [18][19][20][21]. Amphiphilic oligomers of N-vinyl-2-pyrrolidone can be produced by radical telomerization of N-vinyl-2-pyrrolidone in the presence of active chain transfer agents, among which thiols are of particular importance. ...
Development of nanocarrier-based drug delivery systems is a major breakthrough in pharmacology, promising targeted delivery and reduction in drug toxicity. On the cellular level, encapsulation of a drug substantially affects the endocytic processes due to nanocarrier–membrane interaction. In this study we synthesized and characterized nanocarriers assembled from amphiphilic oligomers of N-vinyl-2-pyrrolidone with a terminal thiooctadecyl group (PVP-OD). It was found that the dissolution free energy of PVP-OD depends linearly on the molecular mass of its hydrophilic part up to M¯n = 2 × 104, leading to an exponential dependence of critical aggregation concentration (CAC) on the molar mass. A model hydrophobic compound (DiI dye) was loaded into the nanocarriers and exhibited slow release into the aqueous phase on a scale of 18 h. Cellular uptake of the loaded nanocarriers and that of free DiI were compared in vitro using glioblastoma (U87) and fibroblast (CRL2429) cells. While the uptake of both DiI/PVP-OD nanocarriers and free DiI was inhibited by dynasore, indicating a dynamin-dependent endocytic pathway as a major mechanism, a decrease in the uptake rate of free DiI was observed in the presence of wortmannin. This suggests that while macropinocytosis plays a role in the uptake of low-molecular components, this pathway might be circumvented by incorporation of DiI into nanocarriers.
... In addition, cell nuclei were stained with DAPI nuclear dye (blue fluorescence). It is known that liposomes are quenching fluorescence of compounds incorporated into liposomes [54]. Thus, the fluorescence of labeled drug (paclitaxel) is quenched inside the liposomes. ...
Osimertinib (OSI, AZD9291), is a third-generation, irreversible tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) that selectively inhibits both EGFR-TKI-sensitizing and EGFR T790M resistance mutations. OSI has been approved as a first-line treatment of EGFR-mutant lung cancer and for metastatic EGFR T790M-mutant non-small cell lung cancer. Liposome-based delivery of OSI can provide a new formulation of the drug that can be administered via alternative delivery routes (intravenous, inhalation). In this manuscript, we report for the first time development and characterization of liposomal OSI formulations with diameters of ca. 115 nm. Vesicles were composed of phosphatidylcholines with various saturation and carbon chain lengths, cholesterol and pegylated phosphoethanolamine. Liposomes were loaded with OSI passively, resulting in a drug being dissolved in the phospholipid matrix or actively via remote-loading leading to the formation of OSI precipitate in the liposomal core. Remotely loaded liposomes were characterized by nearly 100% entrapment efficacy and represent a depot of OSI. Passively-loaded vesicles released OSI following the Peppas-Sahlin model, in a mechanism combining drug diffusion and liposome relaxation. OSI-loaded liposomes composed of l-α-phosphatidylcholine (egg-PC) demonstrated a higher toxicity in non-small lung cancer cells with EGFR T790M resistance mutation (H-1975) when compared with free OSI. Developed OSI formulations did not show antiproliferative activity in vitro in healthy lung epithelial cells (MRC-5) without the EGFR mutation.
... These composites have found applications in furniture, packaging, building and automobile industries [10]. Poly(N-vinyl-2-pyrrolidone) (PVP) is widely used in different aspects, such as additives to cosmetics, coatings and biomedicines due to its excellent biocompatibility and hydrophilicity [28][29][30][31][32]. PVP binds to polar molecules exceptionally well owing to its polarity. ...
... The introduction of the demanded terminal group can be achieved by addition of molecular mass regulators to the reaction mixture, their radicals being prone to reinitiation of radical polymerization. The described approach has been used in the synthesis of a series of amphiphilic polymers of N-vinylpyrrolidone [8,9] which can be used as carriers of certain pharmacologically active substances [10,11]. We have earlier described the synthesis of acrylic acid oligomer containing terminal hexylthio group, capable of solubilization of an antituberculous drug (prothionamide) [12]. ...
Synthesis of amphiphilic oligomers of acrylic acid via radical polymerization in the presence of thiols as molecular mass regulators is described. The effect of the length of the introduced hydrophobic terminal group on the critical micellization concentration of the acrylic acid oligomers has been elucidated. It has been shown that the incorporation of an antituberculous drug (prothionamide) in the micelles of acrylic acid oligomers is enhanced with the increase in the length of the hydrophobic moiety. The preparation of hydroxyapatite-filled calcium-polycarboxylate bone cements containing prothionamide (promising for the sealing of postoperative cavities) is described.
... Grafting of PNVP on CHI in aqueous solutions has been reported previously using AIBN as an initiator. [78][79][80][81][82] It has also been used for "grafting from" of PNVP to prepare molecular brushes with pluronic F-127 backbone. 61 Sharma et al. have reported that the grafting of vinyl polymers on CHI with AIBN as an initiator can lead to a polymerization reaction with initiator radical formation at both NH 2 and OH groups of CHI. ...
Hybrid molecular brushes (HMBs) are macromolecules made of a linear backbone and polymeric side chains that differ in their chemical nature. The authors developed a new method of synthesis of HMB with chitosan (CHI) backbone. In the first step, chitosan-graft-polylactide (CHI-g-PLA) was synthesized by interfacial ring opening polymerization of lactide initiated from CHI. CHI-g-PLA is characterized for its molecular weight and structure. In the second step, polyvinylpyrrolidone (PNVP) or polyacrylamide (PAAm) is grafted by radical polymerization from the CHI in CHI-g-PLA to form CHI-g-PLA-g-PNVP and CHI-g-PLA-g-PAAm. This results in the formation of HMB, with hydrophobic PLA and hydrophilic PNVP or PAAm side chains grafted to CHI. The chemical structure and thermal behavior of the HMBs are characterized. The morphology of CHI-g-PLA as well as the HMBs is determined using atomic force microscopy (AFM). Both the HMBs tethered to separate surfaces exhibit reversible switching between the hydrophilic and hydrophobic polymers on exposure to specific solvents. This is studied by AFM and water contact angle measurements. Hence, the authors developed a method for synthesis of HMB that can be applied for surface modification.
... Different moieties can be synthetically anchored to these lipids and introduced to the bilayer to improve their physiochemical properties. A common example is the attachment of hydrophilic polymers as a steric stabiliser, also to reduce liposome recognition and clearance by immune system, such as poly(ethylene glycol) (PEG) [22], poly(vinyl alcohol) [23] and poly(N-vinylpyrrolidones) [24]. This type of liposomes is known as stealth liposomes or long-circulating liposomes. ...
Liposome, one of the most well-established nanomedicines in cancer therapy and bioimaging, is a great delivery system with their flexibility and versatility. Liposomes resemble the biological cell membrane, adopting a lipid bilayer structure which provides protection and solubilisation of both hydrophilic and hydrophobic agents. A wide range of therapeutic drugs and imaging agents can, therefore, be encapsulated and delivered. Cationic liposomes, for example, are one of the popular choices of non-viral vector for the fast-growing field of gene therapy. Their physiochemical properties can be engineered and modified to suit specific applications simply by changing the lipid components and the corresponding ratio. This also expands the potential of having additional functionalities such as long-circulating, targeting and stimuli-responsiveness. In addition to delivering therapeutics and imaging agents, interactions between the lipids and the payloads can be beneficial for imaging enhancement. Stimuli-sensitive liposomes can be used along with diagnostic and therapeutics for image-guided drug delivery, providing real-time monitoring of the drug delivery process as well as spatiotemporal control over the release of drugs. Liposomes will be expected to be a promising delivery system and tool for personalised medicine.
... [14] Typically, the coating of the liposome surface is achieved via incorporation of PEGylated lipid, i.e., the lipid modified with poly(ethylene glycol) (PEG) block, into the liposomal membrane and formation of the "stealth" liposomes. [15][16][17] The liposomal membrane can also be covered by hydrophilic polymers such as poly(vinylpyrrolidone), [18] chitosane, [19] poly(vinyl alcohol), [20] or poly(hydroxypropylmethacrylamide) [21] and its derivatives. [22] Here, we report on the preparation and properties of novel highly stable liposomes, composed of zwitterionic L-αphosphatidylcholine (PC), whose membranes were covered by a hydrophilic N-(2-hydroxypropyl)methacrylamide copolymer containing covalently attached pendant cholesterol fragments (pHPMA). ...
Highly stable liposomes are developed by coating phosphatidylcholine liposomes with amphiphilic N-(2-hydroxypropyl)methacrylamide copolymer. Two approaches in the preparation of coated liposomes are employed: the copolymer is added during (“in prep”) or after (“ex post”) the liposome formation. The influence of polymer concentration and coating method is evaluated using the cryogenic transmission electron microscopy, dynamic light scattering, and small-angle X-ray scattering techniques. The in prep modification significantly increases, up to four weeks, the stability of liposomes against aggregation and makes the liposomal membrane nonpermeable toward an inorganic salt. Such enhanced longevity is attributed to the different structure of in prep coated liposomal membranes.
... Several approaches are now available and efficient for introducing useful functional groups onto PVP. The radical copolymerization of 1-Vinyl-2-pyrrolidinone (VP) with an organic solvent as a radical initiator (e.g., azobisisobutyronitrile or 4,4 -Azobis-(4-cyanovaleric acid), ACVA) and a chain transfer agent (e.g., mercaptoacetic acid or mercaptpropionic acid) is one of the most efficient and effective ways to produce terminal carboxyl-bearing PVP [26][27][28]. This method has for instance allowed for further covalent conjugation of bioactive TNF and IL-6 [27,28], but was ineffective in our study as the synthesized carboxyl-terminated PVP did not allow the formation of silver nanoparticles. ...
![Dieudonné [Radé] Baganizi](https://i1.rgstatic.net/ii/profile.image/279884166385671-1443740993173_Q64/Dieudonne-rade-Baganizi.jpg)
Interleukin-10 (IL-10) is a key anti-inflammatory and immunosuppressive cytokine and therefore represents a potential therapeutic agent especially in inflammatory diseases. However, despite its proven therapeutic efficacy, its short half-life and proteolytic degradation in vivo combined with its low storage stability have limited its therapeutic use. Strategies have been developed to overcome most of these shortcomings, including in particular bioconjugation with stabilizing agents such as polyethylene glycol (PEG) and poly (vinylpyrolidone) (PVP), but so far these have had limited success. In this paper, we present an alternative method consisting of bioconjugating IL-10 to PVP-coated silver nanoparticles (Ag-PVPs) in order to achieve its storage stability by preventing denaturation and to improve its anti-inflammatory efficacy. Silver nanoparticles capped with a carboxylated PVP were produced and further covalently conjugated with IL-10 protein by carbodiimide crosslinker chemistry. The IL-10 conjugated Ag-PVPs exhibited increased stability and anti-inflammatory effectiveness in vitro. This study therefore provides a novel approach to bioconjugating PVP-coated silver nanoparticles with therapeutic proteins, which could be useful in drug delivery and anti-inflammatory therapies.
... The use of pHsensitive peptides or cationic lipids is considered an efficient strategy to enhance the cytoplasmic delivery of liposomal cargo by disrupting the endosome/lysosome membrane following liposomal uptake by phagocytosis and/or endocytosis. [64][65][66][67] Alternatively, incorporation of fusogenic lipids into liposomes and/or the use of cell-penetrating proteins or peptides (CPPs), proteins and peptides that can translocate through the cellular membranes, reportedly may enhance cytoplasmic delivery of liposomal cargo via an endocytosis-independent manner. [68][69][70] However, these non-endocytotic pathways are less prominent 123) than those using endocytosis (Fig. 2). ...
The liposome, a closed phospholipid bilayered vesicular system, has received considerable attention as a pharmaceutical carrier of great potential over the past 30 years. The ability of liposomes to encapsulate both hydrophilic and hydrophobic drugs, coupled with their biocompatibility and biodegradability, make liposomes attractive vehicles in the field of drug delivery. In addition, great technical advances such as remote drug loading, triggered release liposomes, ligand-targeted liposomes, liposomes containing combinations of drugs, and so on, have led to the widespread use of liposomes in diverse areas as delivery vehicles for anti-cancer, bio-active molecules, diagnostics, and therapeutic agents. In this review, we summarize design optimization of liposomal systems and invaluable applications of liposomes as effective delivery systems.
The coating of liposomes with polyethyleneglycol (PEG) has been extensively discussed over the years as a strategy for enhancing the in vivo and in vitro stability of nanostructures, including doxorubicin-loaded liposomes. However, studies have shown some important disadvantages of the PEG molecule as a long-circulation agent, including the immunogenic role of PEG, which limits its clinical use in repeated doses. In this context, hydrophilic molecules as carbohydrates have been proposed as an alternative to coating liposomes. Thus, this work studied the cytotoxicity and preclinical antitumor activity of liposomes coated with a glycosyl triazole glucose (GlcL-DOX) derivative as a potential strategy against breast cancer. The glucose-coating of liposomes enhanced the storage stability compared to PEG-coated liposomes, with the suitable retention of DOX encapsulation. The antitumor activity, using a 4T1 breast cancer mouse model, shows that GlcL-DOX controlled the tumor growth in 58.5% versus 35.3% for PEG-coated liposomes (PegL-DOX). Additionally, in the preliminary analysis of the GlcL-DOX systemic toxicity, the glucose-coating liposomes reduced the body weight loss and hepatotoxicity compared to other DOX-treated groups. Therefore, GlcL-DOX could be a promising alternative for treating breast tumors. Further studies are required to elucidate the complete GlcL-DOX safety profile.
Lipid‐based nanocarriers have demonstrated high interest in delivering genetic material, exemplified by the success of Onpattro and COVID‐19 vaccines. While PEGylation imparts stealth properties, it hampers cellular uptake and endosomal escape, and may trigger adverse reactions like accelerated blood clearance (ABC) and hypersensitivity reactions (HSR). This work highlights the great potential of amphiphilic poly(N‐methyl‐N‐vinylacetamide) (PNMVA) derivatives as alternatives to lipid‐PEG for siRNA delivery. PNMVA compounds with different degrees of polymerization and hydrophobic segments, are synthesized. Among them, DSPE (1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine)‐PNMVA efficiently integrates into lipoplexes and LNP membranes and prevents protein corona formation around these lipid carriers, exhibiting stealth properties comparable to DSPE‐PEG. However, unlike DSPE‐PEG, DSPE‐PNMVA24 shows no adverse impact on lipoplexes cell uptake and endosomal escape. In in vivo study with mice, DSPE‐PNMVA24 lipoplexes demonstrate no liver accumulation, indicating good stealth properties, extended circulation time after a second dose, reduced immunological reaction, and no systemic pro‐inflammatory response. Safety of DSPE‐PNMVA24 is confirmed at the cellular level and in animal models of zebrafish and mice. Overall, DSPE‐PNMVA is an advantageous substitute to DSPE‐PEG for siRNA delivery, offering comparable stealth and toxicity properties while improving efficacy of the lipid‐based carriers by minimizing the dilemma effect and reducing immunological reactions, meaning no ABC or HSR effects.
Numerous nanotech arenas in therapeutic biology have recently provided a scientific platform to manufacture a considerable swath of unique chemical entities focusing on drugs. Recently, nanoparticulate drug delivery systems have emerged to deliver a specific drug to a specified site. Among all other carriers, lipids possess features exclusive to nanostructured dosage forms. The bioavailability of orally administered drugs is typically negatively affected by their poor water solubility, resulting from the unique chemical moieties introduced. Because of their unique advantages, lipid nanoparticles must become increasingly predictable as a robust delivery mechanism. The enhanced biopharmaceutical properties and significance of lipid-based targeting technologies such as liposomes, niosomes, solid-lipid nanoparticles, and micelles are highlighted in this review. Pharmaceutical implications of lipid nanocarriers for the transport and distribution of various therapeutic agents, such as biotechnological products and small pharmaceutical molecules, is a booming topic. Lipid nanoparticles as drug delivery systems have many appealing properties, including high biocompatibility, ease of preparation, tissue specificity, avoidance of reticuloendothelial systems, delayed drug release, scale-up feasibility, nontoxicity, and targeted delivery. The use of lipid nanoparticles to enhance the transport of biopharmaceuticals is currently considered state-of-the-art. Similarly, we critically examine the upcoming guidelines that therapeutic scientists should handle.
Second-generation chiral-substituted poly-N-vinylpyrrolidinones (CSPVPs) (-)-1R and (+)-1S were synthesized by free-radical polymerization of (3aR,6aR)- and (3aS,6aS)-5-ethenyl-tetrahydro-2,2-dimethyl-4H-1,3-dioxolo[4,5-c]pyrrol-4-one, respectively, using thermal and photochemical reactions. They were produced from respective d-isoascorbic acid and d-ribose. In addition, chiral polymer (-)-2 was also synthesized from the polymerization of (S)-3-(methoxymethoxy)-1-vinylpyrrolidin-2-one. Molecular weights of these chiral polymers were measured using HRMS, and the polymer chain tacticity was studied using 13C NMR spectroscopy. Chiral polymers (-)-1R, (+)-1S, and (-)-2 along with poly-N-vinylpyrrolidinone (PVP, MW 40K) were separately used in the stabilization of Cu/Au or Pd/Au nanoclusters. CD spectra of the bimetallic nanoclusters stabilized by (-)-1R and (+)-1S showed close to mirror-imaged CD absorption bands at wavelengths 200-300 nm, revealing that bimetallic nanoclusters' chiroptical responses are derived from chiral polymer-encapsulated nanomaterials. Chemo-, regio-, and stereo-selectivity was found in the catalytic C-H group oxidation reactions of complex bioactive natural products, such as ambroxide, menthofuran, boldine, estrone, dehydroabietylamine, 9-allogibberic acid, and sclareolide, and substituted adamantane molecules, when catalyst Cu/Au (3:1) or Pd/Au (3:1) stabilized by CSPVPs or PVP and oxidant H2O2 or t-BuOOH were applied. Oxidation of (+)-boldine N-oxide 23 using NMO as an oxidant yielded 4,5-dehydroboldine 27, and oxidation of (-)-9-allogibberic acid yielded C6,15 lactone 47 and C6-ketone 48.
Ion pair self-assembly (IPSAM) composed of poly(ethylenimine) (PEI) and (phenylthio)acetic acid (PTA) was prepared to investigate the electric field-responsive release property. PEI/PTA ion pairs were found to have an upper critical solution temperature and were air/water interface-active. IPSAM could release its payload (i.e., nile red) in proportion to the voltage. It was more sensitive to the electric field as PEI/PTA ratio was higher, and more sensitive to temperature change as the voltage was stronger. The electric field-induced protonation and oxidation were thought to be major reasons why IPSAM was responsive to the electric field in terms of release.
The efficacy and safety are two challenging factors before trusting the lipid-based Nanosystems (LBNs) for diagnostic and therapeutic purposes. The aim of this paper is to evaluate the accessible in-vitro and in-vivo studies on the contribution of major physicochemical factors of LBNs for the effective and safe delivery of drugs and anticancer agents into the target site. These factors included lipid charge, particle size and size distribution, lipid composition and components, surface hydrophilicity and hydrophobicity, and surface coating. Here, reliable databases were searched for full-text, accessible and original articles to conduct this review. We included relevant studies since 1992, which were conducted on the development of LBNs for therapeutic and diagnostic purposes. Many studies have shown that using polyethylene glycol modification reduces the undesirable side effects and controls the efficacy and toxicity for in-vitro and in-vivo delivery of drugs or anticancer agents. Also, optimization of the size and composition of lipid nanoparticles minimizes the toxicity of the anticancer agents associated with a carrier. In addition, the presence of positively charged lipids in LBNs improves the efficacy of encapsulated drugs in spite of low blood circulation efficiency and high toxicity effects. The identification of important factors involved in the delivery of LBNs as potential drug carriers is crucial and contributes to the proper design of these interesting carriers for both therapeutic and diagnostic purposes.
Extracellular vesicles (EVs) are carriers of biological signals through export and delivery of RNAs and proteins. Of increasing interest is the use of EVs as a platform for delivery of biomolecules. Preclinical studies have effectively used EVs to treat a number of diseases. Uniquely, endogenous machinery within cells can be manipulated in order to produce desirable loading of cargo within secreted EVs. In order to inform the development of such approaches, an understanding of the cellular mechanisms by which cargo is sorted to EVs is required. Here, the current knowledge of cargo sorting within EVs is reviewed. Here is given an overview of recent bioengineering approaches that leverage these advances. Methods of externally manipulating EV cargo are also discussed. Finally, a perspective on the current challenges of EVs as a drug delivery platform is offered. It is proposed that standardized bioengineering methods for therapeutic EV preparation will be required to create a well‐defined clinical product.
Over the past two decades, RNA interference has become an extensively studied mechanism to silence gene and treat diseases including cancer. siRNA appears as a promising strategy that could avoid some side effects related to traditional chemotherapy. Considering the weak stability of naked siRNA in blood, vectors like cationic liposomes or Lipid Nanoparticles (LNPs) are widely used to carry and protect siRNA until it reaches the tumor targeted. Despite extensive research, only three RNAi drugs are currently approved by the Food and Drug Administration, including only one LNP formulation of siRNA to treat hereditary ATTR amyloidosis. This shows the difficulty of lipoplexes clinical translation, in particular in cancer therapy. To overcome the lipoplexes limitations, searches are made on innovative lipoplexes formulations with enhanced siRNA efficacy. The present review is focusing on the recent use of pH-sensitive lipids, peptides and cell-penetrating peptides or polymers. The incorporation of some of these components in the lipoplex formulation induces a fusogenic property or an enhanced endosomal escape, an enhanced cellular uptake, an enhanced tumor targeting, an improved stability in the blood stream …These innovations appear critical to obtain an efficient siRNA accumulation in tumor cells with effective antitumor effect considering the complex tumor environment.
This book provides a critical overview of the advances being made towards overcoming biological barriers through the contribution of nanosciences and nanotechnologies. Overcoming these barriers is of primary importance for solving the problems of many current drugs and vaccines and it is also especially relevant for the commercial exploitation of new therapeutic strategies i.e. gene and cellular therapies. Despite important information that has been covered in recently published books related to nanomedicine (mainly oriented to technologies and applications), there is still an important gap related to the relationship between the chemical properties of nanobiomaterials and their interaction with the biological environment. Thus, this comprehensive new book that is presented with a clear and organised structure dedicated to the different biological barriers relevant for drug delivery applications will focus on (i) mechanistic issues related to the interaction between drug delivery systems and biological barriers and (ii) the analysis of the critical factors determining the efficacy of nanobiomaterials for specific applications. Moreover, this field has become highly multidisciplinary over the last decade, leading to the development of new products, as well as to significant advances from the regulatory point of view. Moreover, this book will also provide an up-to-date view on the clinical relevance of nanomedicine and on its possible impact on global health.
Polyvinylpyrrolidone (PVP), a non-ionic polymer, has been employed in multifarious fields such as paper, fibers and textiles, ceramics, and pharmaceutics due to its superior properties. Especially in pharmacy, the properties of inertness, non-toxicity, and biocompatibility make it a versatile excipient for both conventional formulations and novel controlled or targeted delivery systems, serving as a binder, coating agent, suspending agent, pore-former, solubilizer, stabilizer, etc. PVP with different molecular weights (MWs) and concentrations is used in a variety of formulations for different purposes. In this review, PVP-related researches mainly in recent 10 years were collected, and its main pharmaceutical applications were summarized as follows: (i) improving the bioavailability and stability of drugs, (ii) improving the physicomechanical properties of preparations, (iii) adjusting the release rate of drugs, and (iv) prolonging the in vivo circulation time of liposomes. Most of these applications could be explained by the viscosity, solubility, hydrophilicity, and hydrogen bond–forming ability of PVP, and the specific action mechanisms for each application were also tried to figure out. The effect of PVP on bioavailability improvement establishes it as a promising polymer in the emerging controlled or targeted formulations, attracting growing interest on it. Therefore, given its irreplaceability and tremendous opportunities for future developments, this review aims to provide an informative reference about current roles of PVP in pharmacy for interested readers.
Aims
This study was designed to prepare chrysophanol-loaded micelles (CLM) to improve the oral bioavailability, targetability and anti-chronic renal failure (CRF) activity of chrysophanol (CH).
Methods
The preparation of CLM was achieved via thin-film dispersion technique. The in vitro release of CLM compared with free CH was measured in phosphate buffer solution (PBS) containing 0.5%w/v sodium dodecyl sulphate (pH 6.8) while the pharmacokinetic and anti-CRF activity study was also conducted in rats. Moreover, the tissue distribution of CLM was investigated in the mice.
Results
The CLM had particle size (PS) of 29.64 ± 0.71 nm, and encapsulation efficiency (EE) of 90.48 ± 1.22%w/w. The cumulative release rate of CH from the micellar system was significantly higher than that of the free CH (86%m/m vs. 15%m/m, p < 0.01). In vivo pharmacokinetic studies showed that the bioavailability of CLM after oral administration was substantially improved (about 3.4 times) compared with free drugs (p < 0.01). Also, it was observed that CLM accumulated well in the liver and brain. Moreover, in vitro renal podocytes study showed that CLM had better protection against renal podocyte damage than the free CH. In addition, CLM significantly (p < 0.01) reduced levels of blood urea nitrogen (BUN), kidney injury molecule-1 (Kim-1), and serum creatinine (SCr), which obviously improved kidney damage in rats with CRF.
Conclusions
Collectively, these findings suggest that mixed micelles may be used as a promising drug delivery system for oral bioavailability improvement and concomitantly enhance the anti-CRF activity of CH, as well as provide a basis for the clinical application of CH.
The possibility of using amphiphilic polymers of N-vinylpyrrolidone as polymer protectors of metal nanoparticles is researched. When metal ions are reduced in aqueous solutions of the corresponding salts in the presence of amphiphilic polymers, copper nanoparticles with a diameter of 4-12 nm or palladium particles with a diameter of 3-5 nm are formed. Nanoparticles are included in rather large aggregates of micelles of amphiphilic polymers. It was found that extremely stable sols are formed in the presence of amphiphilic polymers. In this case, the stability of sols to irreversible aggregation and oxidation of metal nanoparticles increases both with increasing length of the polymer’s hydrophilic chains and with increasing length of hydrophobic fragments. In the presence of a low-molecular-weight electrolyte, particles with a smaller diameter are formed. However, the resistance of metal nanoparticles to oxidation is reduced by 6-10 times.
In this work, we developed a fast, highly efficient, and environmentally friendly catalytic system for classical free-radical polymerization (FRP) utilizing a high-pressure (HP) approach. The application of HP for thermally-induced, bulk FRP of 1-vinyl-2-pyrrolidone (VP) allowed to eliminate the current limitation of ambient-pressure polymerization of ‘less-activated’ monomer (LAM), characterized by the lack of temporal control yielding polymers of unacceptably large disperisites and poor result reproducibility. By a simple manipulation of thermodynamic conditions (p = 125–500 MPa, T = 323–333 K) and reaction composition (two-component system: monomer and low content of thermoinitiator) well-defined poly(1-vinyl-2-pyrrolidone)s (PVP) in a wide range of molecular weights and low/moderate dispersities (Mn = 16.2–280.5 kg mol⁻¹, Đ = 1.27–1.45) have been produced. We have found that HP can act as an ‘external’ controlling factor that warrants the first-order polymerization kinetics for classical FRP, something that was possible so far only for reversible deactivation radical polymerization (RDRP) systems. Importantly, our synthetic strategy adopted for VP FRP enabled us to obtain polymers of very high Mn in a very short time-frame (0.5 h). It has also been confirmed that VP bulk polymerization yields polymers with significantly lower glass transition temperatures (Tg) and different solubility properties in comparison to macromolecules obtained during the solvent-assisted reaction.
Poly(dimethylsiloxane) (PDMS) has been widely used in the field of microfluidics, optical systems, and sensors. However, the hydrophobic nature of PDMS leads to low surface wettability and biofouling problems due to the nonspecific proteins–hydrophobic surface interactions and cell/bacterial adhesion. In this work, the PDMS surface was first introduced with amino groups (PDMS-NH2) via KOH-catalyzed reaction with 3-aminopropyltriethoxysilane (APTES). The PDMS-NH2 was then grafted with poly(N-vinylpyrrolidone) (PVP) based on the self-adhesion reaction between the amino surface and catechol-functionalized PVP (CA-PLL-PVP). CA-PLL-PVP as a comb-polymer was synthesized by conjugating PVP-COOH along with caffeic acid to the ε-polylysine backbone. A significantly enhanced water wettability was observed with contact angles dropped from 116° to 14° after coating with CA-PLL-PVP. The coated surface demonstrated excellent antifouling performance that no appreciable Staphylococcus epidermidis biofilm formation could be observed. This novel facile antifouling coating on PDMS surface may find greater biomedical applications to eliminate the potential adherence problems caused by natural biofouling.
Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA‐pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA‐pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA‐pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA‐pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.
Polymeric derivatives of proteins are synthesized by the reaction of the amino groups of proteins with N,N-diethylacrylamides containing terminal carboxyl groups, having a molecular weight of (13–62) × 10³, and exhibiting a lower critical solution temperature of 34–29°C. It is shown for the first time that the lower critical solution temperature of the obtained derivatives is determined not only by the molecular weight of the polymer and protein but also by the conformation of the protein macromolecule.
This study was designed to investigate the bioavailability and targeting of myricetrin-loaded ternary micelles modified with and without TPGS. The particle diameters of myricetrin-loaded micelles and myricetrin-loaded-TPGS micelle were 30.58 ± 1.34 nm and 26.83 ± 0.89 nm, respectively, while their respective encapsulation efficiencies (m/m, %) were 83.3 ± 1.08 and 93.8 ± 1.18. The release rate of myricetrin in the micellar system clearly exceeded the free myricetrin in the three media (pH 6.8 phosphate buffer, pH 1.2 HCl solution and double distilled water). In vivo studies displayed that the bioavailability of myricetrin mixed micelles was remarkably improved than the free drug after oral administration. Moreover, the results of tissue distribution showed that myricetrin-loaded-TPGS micelles accumulated well in the liver tissue. Based on these results, it was speculated that myricetrin-loaded-TPGS micelles might act as a promising carrier for liver targeting with improved hepatic concentration of myricetrin compared with the myricetrin-loaded micelles.
We report, for the first time, the metal-free green synthesis of linear poly(vinyl pyrrolidone) (PVP) homopolymers of molecular weight higher than 100 kg/mol and narrow dispersities via thermal and photo-induced...
A novel, water soluble, luminescent anthracene-bridged AA-type bi-arm poly(N-vinylpyrrolidone) (ATC-PNVP) has been synthesized using click reaction between alkyne-terminated PNVP and 9,10-bis(azidomethyl)anthracene. The resultant anthracene-bridged PNVP (ATC-PNVP) has been characterized using 1H NMR, FTIR, UV-Vis, fluorescence spectroscopic methods and GPC analysis. ATC-PNVP has shown effective fluorescence properties in aqueous medium. It has shown highly selective “turn off” sensing behaviour towards picric acid, a common nitro-aromatic explosive, with wide linear range of detection of 0.01- 0.3 mM and LOD value of 0.006 mM in water. ATC-PNVP based paper sensors also have shown very effective detection of picric acid within concentration range of 0.001 - 1.0 mM. Effective binding interaction of it with Bovine Serum Albumin (BSA) studied using steady state, synchronous and 3D fluorescence spectroscopy has shown effective quenching of the intrinsic fluorescence of BSA and occurrence of a FRET type interaction. Further, this luminescent ATC-PNVP has been efficiently used as labelling agent for fluorescence microscopy in NIH-3T3 and HeLa cells with greater uptake and hence better fluorescent labelling in cytosol of the tested cell lines as compared to free 9,10-bis (azidomethyl) anthracene. The cell viability study has also shown very good biocompatible and non-toxic nature of ATC-PNVP at lower working concentrations towards all the cell lines tested.
Modification of nanoparticle surfaces with PEG has been widely considered the gold standard for many years. However, PEGylation presents controversial and serious challenges including lack of functionality, hindered cellular interaction, allergic reactions, and stimulation of IgM production after repetitive dosing that accelerates blood clearance of the nanoparticles. We report the development of novel liposomal formulations surface-modified with a low molecular weight, branched polyethyleneimine (bPEI)–lipid conjugate for use as an alternative to PEG. The formulations had very good stability characteristics in ion- and protein-rich mediums. Protein adsorption onto the liposomal surface did not interfere with the cellular interaction. bPEI-modified liposomes (PEIPOS) showed enhanced association with three different cell lines by up to 75 times compared to plain or PEGylated liposomes and were without carrier toxicity. They also penetrated the deeper layers of 3D spheroids. Encapsulating paclitaxel (PTX) into PEIPOS did not change its main mechanism of action. PEIPOS complexed and intracellularly delivered siRNAs and downregulated resistance-associated proteins. Finally, tumor growth inhibition was observed in a mouse ovarian xenograft tumor model, without signs of toxicity, in animals treated with the siRNA/PTX co-loaded formulation. These complex-in-nature but simple-in-design novel liposomal formulations constitute viable and promising alternatives with added functionality to their PEGylated counterparts.
The oligomer of acrylic acid with a thiooctadecyl end-group was obtained by using octadecyl mercaptan as the chain-transfer agent. The resulting oligomer was characterized by ¹ H NMR and ¹³ C NMR spectroscopy and critical micelle concentration was determined in aqueous solution. The order with respect to the initiator concentration was 0.5 and 1.6 with respect to the monomer concentration. The abnormal reaction order with respect to the monomer concentration was explained by participation in the chain propagation of unassociated and associated forms of acrylic acid, which were stabilized by formation of hydrogen bonds. The kinetic parameters of telomerization were determined. Telomerization with acrylic acid in the non-associated form had lower activation energy and lower pre-exponential factor than in the case of associated forms. The synthesis of the acrylic acid oligomer with a thiooctadecyl end-group having a low critical micelle concentration in water was carried out in one stage and corresponds to the concept of atom economy.
Amisulpride (AMS) is an atypical antipsychotic agent used for the treatment of schizophrenia. The effect of different variables, i.e., the type of cyclodextrins (CDs), ratio of drug/CDs, and type of loading on the prepared AMS-CD liposomes (single and double loaded) was studied by applying 23 full factorial design. Double-loaded liposomes are loaded with AMS-hydroxyl propyl-β-cyclodextrin (HP-β-CD) in the aqueous phase and free drug in the lipophilic bilayer, while single-loaded liposomes are loaded only with AMS-HP-β-CD in the aqueous phase. Entrapment efficiency, particle size, polydespersibility, and zeta potential were selected as dependent variables. Design Expert® software was used to obtain an optimized formulation with high entrapment efficiency (64.55 ± 1.27%), average particle size of 40.1 ± 2.77 nm, polydespersibility of 0.44 ± 0.37, and zeta potential of - 48.8 ± 0.28. Optimized formula was evaluated for in vitro release, surface morphology and stability study was also conducted. AMS-HP-β-CD in double-loaded liposomes exhibited higher drug release than those in the conventional liposomes and in the single-loaded liposomes. The maximum plasma concentration (Cmax) of AMS in optimized AMS-HP-β-CD double-loaded liposomal formulation increased by 1.55- and 1.29-fold, as compared to the commercial tablets and conventional liposomes, respectively. However, the relative bioavailability of AMS double-loaded liposomes was 1.94- and 1.28-folds of commercial tablet and conventional liposomes, respectively.
Purpose:
To fabricate an acid-cleavable PEG polymer for the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), and to investigate their ability for endosomal escape and long circulation.
Methods:
PEG-benzaldehyde-hydrazone-cholesteryl hemisuccinate (PEGB-Hz-CHEMS) containing hydrazone and ester bonds was synthesised and used to fabricate a dual pH-sensitive CL-pPSL. Non-cleavable PEGylated pH-sensitive liposome (pPSL) was used as a reference and gemcitabine as a model drug. The cell uptake and endosomal escape were investigated in pancreatic cancer Mia PaCa-2 cells and pharmacokinetics were studied in rats.
Results:
The CL-pPSL showed accelerated drug release at endosomal pH 5.0 compared to pPSL. Compared to pPSL, CL-pPSL released their fluorescent payload to cytosol more efficiently and showed a 1.4-fold increase in intracellular gemcitabine concentration and higher cytotoxicity. In rats, injection of gemcitabine loaded CL-pPSL resulted in a slightly smaller Vd (149 ± 27 ml/kg; 170 ± 30 ml/kg) and shorter terminal T1/2 (5.4 ± 0.3 h; 5.8 ± 0.6 h) (both p > 0.05) but a significantly lower AUC (p < 0.01), than pPSL, due to the lower PEGylation degree (1.7 mol%) which means a 'mushroom' configuration of PEG. A five-time increase in the dose with CL-pPSL resulted in a 11-fold increase in AUC and a longer T1/2 (8.2 ± 0.5 h).
Conclusion:
The PEG-detachment from the CL-pPSL enhanced endosome escape efficiency compared with pPSL, without significantly compromising their stealth abilities.
Molecular self-assembly is a promising strategy for fabrication of nanobiomaterials with remarkable application potential in biomedical fields such as biosensing, bioimaging, tissue engineering, and drug delivery. This chapter focuses on the self-assembly of peptides, drug carriers, and inorganic nanomaterials. The structure, assembly mechanism, and application of these nanobiomaterials are discussed.
Curcumin is the main curcuminoid found in turmeric rizhomes being a strong candidate to formulate foodstuff with specific properties. Among its various bioactive properties, curcumin presents remarkable antiinflammatory activity, however, its low water solubility leads to low absorption. The improvement of its efficacy demands the development of new formulations, and encapsulation is a promising alternative. The objective of the present work was to obtain curcumin-loaded polyvinylpyrrolidone (PVP) nanoparticles and to evaluate the acute in vivo antiinflammatory activity. Nanoparticles were obtained by complexation using the solid dispersion technique and the characterization of the nanoparticles showed that curcumin and PVP formed an amorphous solid solution. Encapsulated curcumin was colloidally stable in distilled water which was attributed to the formation of hydrogen bonds between curcumin hydroxyl and PVP carbonyl groups. Rats were treated orally with single doses of curcumin and curcumin loaded PVP nanoparticles and the antiinflammatory activity was evaluated by an experimental model of carrageenan-induced paw edema, myeloperoxidase (MPO) activity and microcirculation in situ. Treatment with the nanoparticles at 12.5 mg/kg significantly reduced the intensity of edema and MPO activity while pure curcumin only presented significant effect at 400 mg/kg. Curcumin inhibited cell migration since rolling and adherent leukocytes were significantly reduced with nanoparticles at 50 mg/kg and with curcumin at 400 mg/kg. Encapsulated curcumin were effective at lower doses when compared to free curcumin and this may be due to the improved water affinity and colloidal stability of curcumin nanoparticles.
A variety of active pharmaceutical ingredients (APIs) currently used for cancer treatment are cytotoxic, and show nonspecific distribution when administered systemically resulting in toxicity to normal tissues, hence limiting their clinical application. To overcome these challenges, nanocarriers such as liposomes and micelles have been widely used to deliver APIs for cancer chemotherapy. Delivery of nanocarriers is achieved either via “passive targeting” owing to the enhanced permeability and retention (EPR) effect or via “active targeting” due to the presence of various ligands on the surface of nanocarriers, such as antibodies, peptides, etc. Numerous factors are involved in successful delivery of chemotherapeutic agents; these depend on the tumor microenvironment, formulation factors such as choice of ligand use, physiochemical properties of the nanocarriers, and the choice of target. In this chapter, we discuss the fundamentals of EPR effect, factors affecting passive and active targeting, and current clinical update of various actively and passively targeted liposomes and micelles.
Developing new varieties of highly ionic conducting polymer organic–inorganic hybrid ion–gel composites is important for improving the efficiency of energy storage devices to satisfy the current growing need for energy requirements. The scope of iron oxide doped polymer ion–gels in this field has not been investigated much. Therefore, herein, we propose a facile low cost synthesis and material characterization of a new type of flexible, hydrophilic organic–inorganic hybrid ion–gel polymer from polyvinylpyrrolidone (PVP), N-butylpyridinium tetrafluoroborate (bpy[BF4]) ionic liquid (IL) and γ-Fe2O3 magnetic nanoparticles (MNPs) with improved specific capacitance and energy density. The formation of a bpy+˙ cation radical in the thermally stable 1 : 1 PVP–IL ion–gel matrix caused free radical induced thermal polymerisation of PVP chains at higher temperatures giving a flexible PVP–bpy[BF4] ion–gel thin film which was ESR active before and after cross-polymerization. Studies revealed a proton mediated electron migration occurring from PVP to bpy[BF4] causing a reduced rate of electron–hole recombination in the PVP–IL matrix. We have also theoretically probed the intermolecular interactions of PVP and IL by evaluating the PVP–IL binding energy, changes in enthalpy and entropy changes before and after binding, density of states (DOS) distribution and critical points (CP) analysis using Density Functional Theory (DFT). Doping a 1 : 6 wt% ratio of γ-Fe2O3 MNPs into the PVP–IL matrix increased the ionic conductivity of the thin films from 331.7 to 450.66 μS cm−1 and the specific capacitance values from 2.575 and 92.215 F g−1 respectively. The γ-Fe2O3 doped PVP–IL inorganic–organic hybrid thin film was found to be superior compared to the PVP–IL thin film. The high dielectric γ-Fe2O3 MNPs effectively reduced the band gap, electron–hole recombination rate, charge transfer resistance, solid state interface layer resistance and leakage current and also improved the charge–discharge profile and energy density of the newly synthesised PVP–IL ion–gel thin film.
The reaction of the amino group of α-chymotrypsin with poly(N,N-diethylacrylamides) bearing terminal carboxyl groups which have the degree of polymerization ranging from 30 to 180 and which possess an LCST of 34–29°C affords polymer derivatives of the enzyme. It is found that, upon an increase in the temperature of the aqueous solution of the resulting derivatives to 40°C, the derivative with a degree of polymerization of 180 precipitates at 34°C, while the derivatives with a degree of polymerization of 30–80 remain in solution. The activity of α-chymotrypsin as a part of the derivatives with a degree of polymerization of 30 does not change with increasing temperature, whereas the activity of the enzyme as a part of the derivatives with degrees of polymerization of 60 and 80 decays almost to zero near the LCST of the initial polymers. Such a change in the enzyme activity is reversible (the activity fully recovers with a decrease in temperature).
This review discusses the challenges associated with drug delivery and benefits of employing nanosystems in the delivery of small and macromolecular drugs. Poor biopharmaceutical characteristics of drug and biological barriers in the body affect the drug molecules reaching the intended disease site. For instance, solubility and permeability of a drug molecule affect its transport through the cellular membranes, while their stability in the biological environment dictates residence time and efficacy. Nanomedicine, an evaluation of nanotechnology, ferry the payload safely and effectively through several anatomical and physiological barriers to the target site. Besides, nanomedicine could be engineered to provide compound effect through ligand-mediated targeting and image guided drug delivery at disease site. With illustrative examples from scientific literature, the versatility of different nanosystems and their utility in disease therapy spanning from preclinical development to approved products is emphasized. Specific issues in drug approval including quality-by-design and regulatory aspects are discussed. Based on the advances in drug delivery and nanomaterial synthesis, there is a great future for nanomedicine in diagnosis and treatment of several complex diseases.
Amphiphilic N-vinylpyrrolidone polymers were prepared by a new one-step procedure consisting in radical polymerization of the monomer in the presence of long-chain monobasic saturated carboxylic acid chlorides as chain-transfer and chain-terminating agents. The behavior of the new amphiphilic polymers differing in the structure of the hydrophilic and hydrophobic moieties in aqueous media was studied. The synthesized polymers at definite concentrations undergo spontaneous aggregation with the formation of spherical nanosized micellar particles consisting of a hydrophobic core and a hydrophilic shell. The main characteristics of the polymer nanoparticles formed were determined, and the possibility of using them as promising carriers for the delivery of biologically active compounds and drugs was revealed.
The primary purpose of the present study was to design and optimize a liposomal formulation of the poorly water-soluble drug oleanolic acid (OA) to improve its oral bioavailability, and prolong the duration of therapeutic drug level. Liposomes containing a soybean lecithin and cholesterol lipid bilayer, a protective hydrophilic polyvinylpyrrolidone-K30 (PVP-K30) coating, and a protective bile salt, sodium deoxycholate, were prepared by a thin-film dispersion method coupled with sonication. Several properties of the PVP-modified OA liposomes (PVPOALs), including surface morphology, particle size, zeta potential and entrapment efficiency were extensively characterized. The pharmacokinetic parameters of PVPOALs in rats were determined by UPLC-MS/MS following oral administration. The results of the characterization studies demonstrated that PVPOALs were spherical particles with an average particle size of 179.4 nm and a zeta potential of −28.8 mV. The drug encapsulation efficiency was more than 90%. After freeze-drying, the prepared liposomes possessed high entrapment efficiency of more than 90%. The mean particle size was 194.8 nm, and the zeta potential was about −30.9 mV. Furthermore, as compared to the commercial tablets, the liposomal formulation enhanced the maximum plasma concentration (Cmax) of OA by 6.90-fold in rat plasma. The relative bioavailability of PVP-modified liposomes was 607.9%. The research work in the paper suggests that PVP-modified liposomes could serve as a practical oral preparation for OA in future cancer therapy.
New therapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL.
The current treatment of leishmaniasis has been hampered due to the high toxicity of the available drugs and long duration protocols, which often lead to its abandonment. In the present study, a poloxamer 407-based delivery system was developed, and a molecule, 8-hydroxyquinoline (8-HQN), was incorporated with it, leading to an 8-HQN/micelle (8-HQN/M) composition. Assays were performed to evaluate the in vitro antileishmanial activity of 8-HQN/M against Leishmania amazonensis stationary promastigotes. The cytotoxicity in murine macrophages and in human red cells, as well as the efficacy of the treatment in macrophages infected by parasites, was also assessed. This product was also evaluated for the treatment of murine tegumentary leishmaniasis, using L. amazonensis-infected BALB/c mice. To evaluate the in vivo efficacy of the treatment, the average lesion diameter (area) in the infected tissue, as well as the parasite load at the site of infection (skin), spleen, liver and draining lymph nodes were examined. Non-incorporated micelle (B-8-HQN/M) and the free molecule (8-HQN) were used as controls, besides animals that received only saline. The parasite burden was evaluated by limiting dilution and quantitative real-time PCR (qPCR) techniques, and immunological parameters associated with the treatments were also investigated. In the results, the 8-HQN/M group, when compared to the others, presented more significant reductions in the average lesion diameter and in the parasite burden in the skin and all evaluated organs. These animals also showed significantly higher levels of parasite-specific IFN-γ, IL-12, and GM-CSF, associated with low levels of IL-4 and IL-10, when compared to the saline, 8-HQN/M, and B-8-HQN groups. A predominant IL-12-driven IFN-γ production, against parasite proteins, mainly produced by CD4+ T cells, was observed in the treated animals, post-infection. In conclusion, 8-HQN/M was highly effective in treating L. amazonensis-infected BALB/c mice and can be considered alone, or combined with other drugs, as an alternative treatment for tegumentary leishmaniasis.
Graphical Abstract
Therapeutic scheme and immunological and parasitological parameters developed in the present study
Protein- and peptide-based drugs are currently among the most effective treatment options for various diseases and conditions including cancer, diabetes and hemophilia. To successfully deliver peptides and proteins to their site of action, two main obstacles have to be overcome. First, the peptide and protein therapeutics need to be protected until they reach their site of action; second they need to be delivered intracellularly into the cells. This chapter focuses on the intracellular delivery of peptides and proteins. Successful noninvasive peptide and protein delivery depends on nontoxic carriers or vectors, which can efficiently deliver the macromolecular drug intracellularly to exert their therapeutic action inside the cytoplasm or onto nucleus or other specific organelles, such as lysosomes, mitochondria, or endoplasmic reticulum. The chapter then discusses transferrin receptor-mediated delivery and folate receptor-mediated delivery of peptides and proteins. It also explains transmembrane delivery of peptides and proteins.
Currently used pharmaceutical nanocarriers, such as liposomes, micelles, nanoemulsions, polymeric nanoparticles and many others demonstrate a broad variety of useful properties, such as longevity in the blood allowing for their accumulation in pathological areas with compromised vasculature; specific targeting to certain disease sites due to various targeting ligands attached to the surface of the nanocarriers; enhanced intracellular penetration with the help of surface-attahced cell-penetrating molecules; contrast properties due to the carrier loading with various contrast materials allowing for direct carrier visualization in vivo; stimuli-sensitivity allowing for drug release from the carriers under certain physiological conditions, and others. Some of those pharmaceutical carriers have already made their way into clinic, while others are still under preclinical development. What could be seen much more rare, however, are the pharmaceutical nanocarriers combining several from the listed abilities. Long-circulating immunoliposomes capable of prolonged residence in the blood and specific target recognition represent one of few examples of this kind. At the same time, the enginnering of multifunctional pharmaceutical nanocarriers combinig several useful preoperties in one particle can significantly enhance the efficacy of many therapeutic and diagnostic protocols. This paper considers current status and possible future directions in the emerging area of multifunctional nanocarriers with primary attention on the combination of such properties as longevity, targetability, intracellular penetration and contrast loading.
Poly(N-vinyl pyrrolidone)-block-poly(N-vinyl carbazole)-block-poly(N-vinyl pyrrolidone) (PVP-b-PVK-b-PVP) triblock copolymers were synthesized via sequential reversible addition-fragmentation chain transfer/macromolecular design via the interchange of xanthate (RAFT/MADIX) process. First, 1,4-phenylenebis(methylene)bis(ethyl xanthate) was used as a chain transfer agent to mediate the radical polymerization of N-vinyl carbazole (NVK). It was found that the polymerization was in a controlled and living manner. Second, one of α,ω-dixanthate-terminated PVKs was used as the macromolecular chain transfer agent to mediate the radical polymerization of N-vinyl pyrrolidone (NVP) to obtain the triblock copolymers with various lengths of PVP blocks. Transmission electron microscopy (TEM) showed that the triblock copolymers in bulks were microphase-separated and that PVK blocks were self-organized into cylindrical microdomains, depending on the lengths of PVP blocks. In aqueous solutions, all these triblock copolymers can self-assemble into the spherical micelles. The critical micelle concentrations of the triblock copolymers were determined without external adding fluorescence probe. By analyzing the change in fluorescence intensity as functions of the concentration, it was judged that the onset of micellization occurred at the concentration while the FL intensity began negatively to deviate from the initial linear increase with the concentration. Fluorescence spectroscopy indicates that the self-assembled nanoobjects of the PVP-b-PVK-b-PVP triblock copolymers in water were capable of emitting blue/or purple fluorescence under the irradiation of ultraviolet light. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016
The proteins associated with liposomes in the circulation of mice were analyzed in order to determine whether bound proteins significantly influence the fate of liposomes in vivo. Liposomes were administered intravenously via the dorsal tail vein of CD1 mice and were isolated from blood after 2 min in the absence of coagulation inhibitors using a rapid "spin column" procedure. Various negatively charged liposomes exhibiting markedly different clearance properties were studied; notably, these included liposomes containing 10 mol % ganglioside GM1 which has been previously shown to effectively limit liposomal uptake by the fixed macrophages of the reticuloendothelial system. The protein binding ability (PB; g of protein/mol of lipid) of the liposomes was quantitated and related to the circulation half-life (tau 1/2) of the liposomes. Liposomes having similar membrane surface charge imparted by different anionic phospholipids were found to exhibit markedly different protein binding potentials. Furthermore, PB values determined from the in vivo experiments were found to be inversely related to circulation half-lives. PB values in excess of 50 g of protein/mol of lipid were observed for rapidly cleared liposomes such as those containing cardiolipin or phosphatidic acid (tau 1/2 less than 2 min). PB values for ganglioside GM1-containing liposomes (tau 1/2 greater than 2 h) were significantly less (PB less than 15 g of total protein/mol of total lipid). PB values were also determined for liposomes recovered from in vitro incubations with isolated human serum; relative PB values obtained from these in vitro experiments were in agreement with relative PB values measured from in vivo experiments. PB values, therefore, could be a useful parameter for predicting the clearance behavior of liposomes in the circulation. Liposomes exhibiting increased PB values in vivo were shown by immunoblot analysis to bind more immune opsonins, leading to a higher probability of phagocytic uptake. Finally, based on results obtained using the in vitro system, it is suggested that the mechanism by which ganglioside GM1 prolongs the murine circulation half-life of liposomes is by reducing the total amount of blood protein bound to the liposomes in a relatively nonspecific manner.
The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.
Injectable nanoparticulate carriers have important potential applications such as site-specific drug delivery or medical imaging.
Conventional carriers, however, cannot generally be used because they are eliminated by the reticulo-endothelial system within
seconds or minutes after intravenous injection. To address these limitations, monodisperse biodegradable nanospheres were
developed from amphiphilic copolymers composed of two biocompatible blocks. The nanospheres exhibited dramatically increased
blood circulation times and reduced liver accumulation in mice. Furthermore, they entrapped up to 45 percent by weight of
the drug in the dense core in a one-step procedure and could be freeze-dried and easily redispersed without additives in aqueous
solutions.
In preclinical studies, a doxorubicin liposome formulation containing polyethylene-glycol (Doxil) shows a long circulation time in plasma, enhanced accumulation in murine tumors, and a superior therapeutic activity over free (unencapsulated) doxorubicin (DOX). The purpose of this study was to characterize the pharmacokinetics of Doxil in cancer patients in comparison with free DOX and examine its accumulation in malignant effusions. The pharmacokinetics of doxorubicin and/or liposome-associated doxorubicin were analyzed in seven patients after injections of equivalent doses of free DOX and Doxil and in an additional group of nine patients after injection of Doxil only. Two dose levels were examined, 25 and 50 mg/m2. When possible, drug levels were also measured in malignant effusions. The plasma elimination of Doxil followed a biexponential curve with half-lives of 2 and 45 h (median values), most of the dose being cleared from plasma under the longer half-life. Nearly 100% of the drug detected in plasma after Doxil injection was in liposome-encapsulated form. A slow plasma clearance (0.1 liter/h for Doxil versus 45 liters/h for free DOX) and a small volume of distribution (4 liters for Doxil versus 254 liters for free DOX) are characteristic of Doxil. Doxorubicin metabolites were detected in the urine of Doxil-treated patients with a pattern similar to that reported for free DOX, although the overall urinary excretion of drug and metabolites was significantly reduced. Doxil treatment resulted in a 4- to 16-fold enhancement of drug levels in malignant effusions, peaking between 3 to 7 days after injection. Stomatitis related to Doxil occurred in 5 of 15 evaluable patients and appears to be the most significant side effect in heavily pretreated patients. The results of this study are consistent with preclinical findings indicating that the pharmacokinetics of doxorubicin are drastically altered using Doxil and follow a pattern dictated by the liposome carrier. The enhanced drug accumulation in malignant effusions is apparently related to liposome longevity in circulation. Further clinical investigation is needed to establish the relevance of these findings with regard to the ability of liposomes to modify the delivery of doxorubicin to solid tumors and its pattern of antitumor activity.
Protective effect of poly(ethylene glycol) and some other polymers on liposomes is considered in terms of statistical behavior of macromolecules in solution. According to the mechanism proposed, liposome-grafted chains of flexible and hydrophilic polymers form dense “conformational clouds” preventing other macromolecules from the surrounding solution from interaction with the liposome surface even at low concentration of protecting polymer. The scale of protective effect is interpreted as the balance between the energy of hydrophobic anchor interaction with the membrane core and the energy of polymer chain free motion in solution. Experimental evidences are presented for the importance of protecting polymer flexibility in preventing liposome opsonization, and possible further theoretical applications of the suggested model are discussed. The possibility of using protecting polymers other than poly(ethylene glycol) is analyzed, and examples of such polymers are given basing on polymer-coated liposome biodistribution data.
A study has been made of the effect of mercaptoesters of the general formula RCOOCH2CH2SH (where R is a hydrocarbon radical containing four to fourteen carbon atoms) on the rate of polymerization of methyl methacrylate and methacrylic acid. It was found that addition of small quantities of mercaptoesters to the original monomer composition results in increase in the steady state polymerization rate in homogeneous and heterogeneous systems. It is suggested that the reason for this increase in rate is a “formal” increase in the efficiency of initiation, resulting from the fact that in the presence of a mercaptoester reaction of the primary radicals from the initiator with the chain-transfer agent competes with recombination of these radicals. A greater increase in the rate of polymerization of methacrylic acid is evidently related to the heterogeneity of the system. A reduction in the rate of both homogeneous and heterogeneous polymerization when the quantity of regulator is increased is evidently explained by increase in the part played by reaction of the growing radical with a radical of the transfer agent and by dimerization of the transfer-agent radicals. A shift of the gel effect to higher degrees of conversion in polymerization of methyl methacrylate in the presence of mercaptoesters is evidently because the viscosity of the system necessary to cause the gel effect is reached at higher degrees of conversion as a result of the reduction in molecular weight in the presence of the regulator.
The protective effect of poly(ethylene glycol) and some other polymers on nanoparticulate carriers including liposomes is considered in terms of statistical behavior of macromolecules in solution, when polymer flexibility plays a key role. According to the mechanism proposed, surface-grafted chains of flexible and hydrophilic polymers form dense “conformational clouds” preventing other macromolecules from the interaction with the surface even at low concentration of protecting polymer. Using liposomes as an example, experimental evidence is presented of the importance of protecting polymer flexibility in liposome steric protection. Further possible applications of the suggested model are discussed. The possibility of using protecting polymers other than poly(ethylene glycol) is analyzed, and examples of such polymers are given based on polymer-coated liposome biodistribution data. General requirements for protecting polymers are formulated, and differences in steric protection of liposomes and particles are discussed. The scale of protective effect is interpreted as the balance between the energy of hydrophobic anchor interaction with the liposome membrane core or with the particle surface and the energy of polymer chain free motion in solution.
The esterification of a carboxy group terminated vinylpyrrolidone oligomer with chloromethylstyrene under mild conditions resulted in a styryl terminated oligovinylpyrrolidone macromonomer. The purity of the obtained macromonomer was greater than 95%. Copolymerization of the macromonomer with styrene in ethanol using BPO as catalyst yielded a graft copolymer having a hydrophobic backbone and amphiphilic branches, which was found to have a spherical form and was dispersable in water and ethanol.Die Veresterung eines carboxygruppenterminierten Vinylpyrrolidon-Oligomeren mit Chlormethylstyrol unter milden Bedingungen führte zu einem styrylterminierten Oligovinylpyrrolidon-Makromonomeren. Die Reinheit des erhaltenen Makromonomeren war größer als 95%. Die Copolymerisation des Makromonomeren mit Styrol in Ethanol mit BPO als Katalysator ergab ein Pfropfcopolymeres mit hydrophobem Rückgrat und amphiphilen Seitenketten, das eine kugelförmige Gestalt hatte und dispergierbar in Wasser und Ethanol war.
Association of drugs with carriers such as liposomes has marked effects on both the pharmacokinetic profiles of the carrier and of the carrier-associated drug. In general, association of drugs with liposomes delays drug absorption, alters and restricts drug biodistribution, decreases the volume of distribution, delays clearance and retards drug metabolism. Surface modification of liposomes by the inclusion of hydrophilic components (e.g., carbohydrates, glycolipids or polymers) to form long-circulating liposomes causes changes in the pharmacokinetic pattern seen for unmodified (classical) liposomes. While classical liposomes have non-linear, saturable kinetics, long-circulating liposomes possess dose-independent, non-saturable, log-linear kinetics. The log-linear kinetics for long-circulating liposomes results from a significant decrease in the first phase of clearance into a high affinity, low capacity system, probably the cells of the mononuclear phagocyte system. An understanding of the pharmacokinetics of liposome-associated drugs is critical to the development of rationale strategies for therapeutic applications of long-circulating liposomes.
Polyethylene glycol (PEG) is widely used as a covalent modifier of biological macromolecules and particulates as well as a carrier for low molecular weight drugs. In the first two instances proteins and liposomes are of particular importance. Their conjugates with PEG often possess the ability to avoid quick recognition and clearance in vivo, that their unconjugated counterparts are suffering from. In this review (with 133 references) methods for preparation of PEG conjugates with various biologically active compounds are summarized. Since the bulk of the published work in this field involves proteins, drugs, and lipids, an appropriate emphasis is given to the conjugates of these compounds. While the first two types of PEG conjugates are usually intended for a direct use as therapeutics, PEG-lipids are mainly utilized for formation of long-circulating liposomes. Particular attention is paid to the comparative attributes of various reactive PEG derivatives, properties of the linkages formed, and possible side reactions. The relationships between various conjugation strategies and their influence on the relevant biological properties and/or on in vivo performance of the corresponding conjugates is also discussed.
The pharmacokinetics and biodistribution of liposome-encapsulated drugs are controlled by the interplay of two variables: the rate of plasma clearance of the liposome carrier, and the stability of the liposome-drug association in circulation. Inhibition of the rapid uptake of liposomes by the reticuloendothelial system and reduction of the rate of drug leakage have resulted in long-circulating liposomal drug systems with valuable pharmacologic properties. These carrier systems show an improved extravasation profile with enhanced localization in tumors and possibly in other tissues, such as skin. An anticancer drug, doxorubicin, encapsulated in polyethyleneglycol-coated, long-circulating liposomes, shows a unique pharmacokinetic/toxicity pattern and promising antitumor activity in initial clinical studies.
An important, if not the chief, condition for the prolongation of the circulation times of lipid vesicles in vivo is the suppression of macromolecular adsorption onto the surface of such vesicles. This adsorption can be prevented very efficiently by a zone of suitably designed and mobile steric hindrances near the lipid layer surface. Lipid vesicles with such a surface coat, cryptosomes, thus circulate in blood for very long periods of time after systemic applications. Lipid vesicles composed of phosphatidylcholine molecules and of suitable polyoxyethylene (PEG) derivatives of phosphatidylethanolamine, for example, remain in the blood circulation 8–10-times longer than standard liposomes made of phosphatidylcholine only: in mice the half-lifes of the former and latter vesicles, after an i.v. administration, are approx. 0.6 h and between 5.9 and 13.8 h, respectively. Vesicle longevity is not destroyed by the phosphatidylcholine chains fluidity. Vesicles consisting of a mixture of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) with distearoylphosphatidylcholine or cryptosomes made of DSPE-PEG and soy-bean phosphatidylcholine, consequently, have a very similar fate in vivo. Furthermore, the cryptosome longevity is not affected directly by the presence of the net charges on the lipid membranes and is little sensitive to the details of the group coupling the PEG-headgroups and the lipidic (hydrophobic) anchor. However, the life-time and the distribution of the stabilized lipid vesicles in vivo depend quite sensitively on the surface density of the sterically active headgroups; often (if not always) the resistance to plasma components adsorption as well as the resulting longevity in vivo both show a maximum near the same lipid/stabilizer molar ratio. Optimum bilayer composition may differ for the different combinations of the main and sterically active membrane components. Its position is probably determined by the variations in the molecular mobility and the effective surface-coverage effects: both must be sufficiently high for the vesicle phagocytosis and accumulation in the reticulohystiocytic system to be suppressed. On the contrary, the bilayer surface hydrophilicity, which hitherto has been believed to be of paramount importance for the liposome longevity in vivo, is per se not relevant for the biological fate of the lipid vesicles, provided that this hydrophilicity exceeds some minimum value.
This paper explores the mechanism(s) whereby liposomes accumulate in chronically ischaemic myocardium and intestine. Plasma prepared from venous blood obtained at sites of myocardial and intestinal infarction does not promote the lysis of positively and negatively charged liposomes in vitro. Albumin-bound lysophosphatidylcholine (≥ 2 mM) lyses positively and negatively charged liposomes in vitro at similar rates. [99mTc]Diethylenetriamine pentaacetic acid (DTPA) entrapped in positively charged liposomes was accumulated in ischaemic rat caecum/colon 6 and 24 h after mesenteric ligation. Presumably allied to the accumulation of liposomal components, necrotic caecum/colon showed marked Ca2+ accumulation and phospholipid depletion. It is postulated that Ca2+ and Ca2+-activated membrane phospholipases may be implicated in the mechanism of liposome accumulation in chronic ischaemia.
Biodistribution and infarct accumulation of different liposome preparations in rabbits with experimental myocardial infarction have been investigated. The influence of such parameters as liposome size, and presence or absence of poly(ethylene glycol) (PEG) and infarct-specific antimyosin antibody (AM) on liposome behavior in vivo was studied. All three variables were shown to affect liposome biodistribution, liposome size being the least significant variable. Statistical analysis of the data obtained demonstrated that of all variables, PEG coating expresses the strongest influence on the liposome blood clearance, significantly (P = 0.0001) increasing the mean level of blood radioactivity under all circumstances. Infarct accumulation depended upon the presence of both PEG (P = 0.0013) and AM (P = 0.005). The infarct-to-normal ratio was affected by the presence of AM (P = 0.0002), but the extent of the effect depended also on the presence of PEG (P = 0.01). Two differing mechanisms can be seen in infarct accumulation of PEG-liposomes (slow accumulation via the impaired filtration) and AM-liposomes (specific binding of immunoliposomes with the exposed antigen). Both mechanisms are supplementary in case of liposomes carrying PEG and AM at the same time. An optimization strategy is suggested for using liposomes as carriers for diagnostic (a high target-to-nontarget ratio is required) and therapeutic (a high absolute accumulation in the target is required) agents.
Sterically stabilized (Stealth®) liposomes, in which the surface of liposomes has been altered, have been developed through the use of glycolipids (principally monosialoganglioside GM1) or polymers (principally polyethylene glycol). The resulting surface is more hydrophilic, and less able to bind opsonins from plasma. This results in liposomes which have decreased uptake into the mononuclear phagocyte system (MPS), increased circulation half-lives, increased stability to contents leakage, and dose-independent pharmacokinetics. These are significant advantages compared to conventional liposome formulations for use of liposomes in a number of therapeutic applications.
To obtain liposomes with longer circulation times in vivo, we newly synthetized dipalmitoylphosphatidylpolyglycerol (DPP-PG). A series of DPP-PGs of different chain lengths was used in this study. The individual derivatives were incorporated into distearoylphosphatidylcholine/cholesterol liposomes (1:1, molar ratio). The effectiveness of DPP-PG derivatives was dependent on the amount and degree of polymerization. Low-polymerized PGs such as diglycerol and tetraglycerol needed a high incorporation rate of 8 mol%, while high-polymerized PGs such as octaglycerol required 4 mol%. The incorporation of 6 mol% of DPP-hexaglycerol was most effective in prolonging the circulation time of liposomes.
The effect of negative surface charge and hydrophilic groups on liposome clearance from blood was investigated in mice using liposome-entrapped 67gallium-deferoxamine as a label. The presence of negatively-charged lipids may retard or accelerate liposome clearance. Physicochemical features contributing to optimal retardation of liposome clearance include a hydrophilic carbohydrate moiety and a sterically hindered negatively-charged group. The relevance of the negative charge steric effect is suggested by the finding that phosphatidylinositol phosphate (PIP) and trisialoganglioside (GT1) are less effective than phosphatidylinositol (PI) and monosialoganglioside (GM1), respectively, in retarding liposome clearance. The need for negative charge in addition to the carbohydrate group for optimal effect on retardation of clearance is indicated by the observation that asialoganglioside (AGM1) is less effective than GM1 in this respect. The negative charge effect is observed with liposome bilayers having both low and high temperature phase-transitions. Increasing the molar fraction of negatively-charged lipid (hydrogenated PI derived from soya) from 23 to 41% resulted in a dramatic acceleration of liposome clearance. The clearance-accelerating effect of the high negative charge was specifically directed to the liver with selective reduction of spleen uptake. Increasing liposome size also had an accelerating effect on clearance but in this case it was accompanied by a non-specific concomitant increase of both liver and spleen uptake.
Removal of intravenously injected liposomes from the circulation is achieved by cells of the mononuclear phagocyte system, also known as the reticuloendothelial system. On exposure to blood, liposomes become coated with plasma proteins; some of these proteins (opsonins) are thought to determine their recognition by mononuclear phagocytes. This review provides a critical discussion of factors that control opsonization of liposomes and their phagocytosis in vivo and in vitro.
Newly developed liposomes with prolonged circulation half-lives and dose-independent pharmacokinetics (Stealth liposomes) have been tested for their efficacy as a slow release system for the rapidly degraded, schedule-dependent, antineoplastic drug 1-beta-D-arabinofuranosylcytosine (ara-C) in the treatment of murine L1210/C2 leukemia. Mice were given injections of either 10(5) cells or 10(6) cells by either the i.v. or the i.p. routes. Leukemia-bearing mice were treated with either i.v. or i.p. injections of free drug, i.v. or i.p. injections of liposome-entrapped drug, or 24-h i.v. infusions of free drug. Long-circulating liposomes contained, as the stealth component, either monosialoganglioside or polyethylene glycol-distearoylphosphatidylethanolamine. Liposomes lacking the stealth components (non-stealth liposomes) were also injected for comparison. At lower dose ranges, stealth liposomes were superior to non-stealth liposomes in prolonging mean survival times of the mice, and all liposome preparations were superior to injections of the free drug. Drug entrapped in stealth liposomes, when administered at or near the maximum tolerated dose of 100 mg/kg ara-C were considerably superior to 24-h free drug infusions given at the same total drug dose. Therapeutic effect was related to the half-life of leakage of ara-C from the liposome formulations, as well as to circulation half-life, with maximum therapeutic effect achieved with long circulation half-lives and more rapid leakage rates. The therapeutic efficacy of non-stealth liposomes increased with increasing liposome (and drug) dose as a result of saturation of liposome uptake by the mononuclear phagocyte system, which resulted in longer circulation half-lives for these liposomes at higher doses (Michaelis-Menten pharmacokinetics). Liposome entrapment can protect rapidly degraded drugs from breakdown in vivo, with release of the drugs in a therapeutically active form over periods of up to several days. The dose-independent pharmacokinetics and reduced mononuclear phagocyte system uptake of stealth liposomes gives them distinct advantages over non-stealth liposomes.
Liposome membranes containing lipids with covalently attached poly(ethylene glycol) (PEG-lipid) are currently being developed as drug delivery systems. These, so called, 'Stealth' liposomes have a relatively long half life (approximately 1 day) in blood circulation and show an altered biodistribution in vivo. The extended lifetime appears to result from a steric stabilization of the liposome by the grafted polymer. In order to characterize the surface structures that promote steric stability in such polymer-grafted lipid bilayer systems, we have used X-ray diffraction to measure the structural organization and interbilayer repulsion for lipid/cholesterol (2:1) bilayers incorporating 4 mol% of a PEG-lipid in which the molecular weight of the PEG moiety was 1900 g/mol. At this concentration, applied pressure versus interbilayer distance relations showed that the grafted polymer moiety extended approximately 50 A from the lipid surface and gave rise to a strong, slowly decaying repulsive pressure between membranes that opposed their close approach. Also, the pressure vs. distance relations were only modestly altered by changing the ionic strength of the medium (1 mM NaCl and 100 mM NaCl). Therefore, even though the PEG-lipid headgroup bears a negative charge, the long range pressure cannot be due primarily to an electrostatic double layer pressure. Measurements of lipid bilayer elasticity using micropipet manipulation showed that PEG-lipid did not change the cohesive properties of lipid/cholesterol liposomes which was consistent with the X-ray structural data showing that the PEG-lipid did not change the normal structure of the bilayer interior. From these data we conclude that the repulsive barrier properties of lipid-grafted PEG polymer chains originate mainly from a steric pressure and that this simple polymer steric stabilization is the basis for the extended in vivo circulation times observed for polymer-grafted liposomes.
Sterically stabilized polyethylene oxide-polystyrene copolymer microspheres, (PS-PEO) and charge stabilized polystyrene (PS) microspheres of similar size (1 micron) were prepared in order to compare their uptake by cultured rat Kupffer cells isolated by centrifugal elutriation. The uptake of the sterically stabilized particles was found to be much less than that for the charge stabilized control. The uptake of microspheres stabilized with covalently grafted PEO was lower or equivalent to that of control microspheres stabilized by the adsorption of the non-ionic PEO-polypropylene oxide (PPO-PEO) surfactant Poloxamer 238 or Methoxy-PEO. Phagocytic uptake by Kupffer cells at low and body temperature (8 degrees C and 37 degrees C) demonstrated that PS-PEO particles showed both low adherence and low metabolic uptake. The adsorption of PEO, as Poloxamer 238, to particles with covalently attached or grafted PEO resulted in a synergistic reduction in uptake that was greater than the individual effects of grafting and adsorption alone (P less than or equal to 0.001). It is suggested that this combination produces a more effective steric barrier on the particle surface with the Poloxamer adsorbing to the surface between the grafted PEO chains. The relevance to drug targeting/carrier systems is discussed.
In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo.
Well-defined liposome systems have previously established the influence of size, surface charge lipid composition and surface ligands, on in vivo fate and behaviour of model compounds entrapped in liposomes. In the present study, preformed liposomes which quantitatively retain aqueous markers were covalenty coupled via dipalmitoylphosphatidyl-ethanolamine, to the hydrophilic polymer, monomethoxypoly(ethylene glycol) (MPEG 5000). Such liposomes retain the coating in the presence of plasma, and appear to adsorb plasma components more slowly than liposomes without the polymer, shown using an aqueous two-phase partitioning technique. MPEG-coupled liposomes were cleared from the blood circulation up to 30% more slowly than liposomes without MPEG after intravenous administration to mice, despite the unmodified liposomes being of a composition and size shown previously to favour achievement of maximum half-life. It is suggested that the polymer acts as a surface barrier to plasma factors which otherwise bind to liposomes in the blood and accelerate vesicle removal.
A series of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) of different polymer chain length was used in this study. Both the activity of PEG-PE in prolonging the circulation time of liposomes and the relative steric barrier activity of amphipathic polymer, measured by a liposome agglutination assay, were found to be directly proportional to the chain length of PEG-PE (PEG5000-PE greater than PEG2000-PE greater than PEG750-PE). However, PEG5000-PE caused a reduced target binding of immunoliposomes in mice due to its overly strong steric barrier activity. The best PEG-PE species supporting the target binding of immunoliposomes was PEG2000-PE, the activity of which was compatible to that of ganglioside GM1. However, GM1 only showed a weak steric barrier activity, suggesting a different mechanism for this glycolipid.
Incorporation of dioleoyl N-(monomethoxy polyethyleneglycol succinyl)phosphatidylethanolamine (PEG-PE) into large unilamellar liposomes composed of egg phosphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37 degrees C, yet the liposomes show a significant increase in the blood circulation half-life (t1/2 = 5 h) as compared to those without PEG-PE(t1/2 less than 30 min). The PEG-PE's activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, a well-described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that of GM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.
Of fundamental importance in the design of a therapeutic drug carrier system is a thorough understanding of the factors which control its fate in the living animal. The use of liposomes as a carrier system able to improve the therapeutic efficacy of a wide range of drugs, requires manipulation of its physical characteristics, thereby influencing in vivo behavior. This review brings together findings of recent studies which describe how liposomal stability and clearance in vivo are controlled by the architecture of the vesicles themselves which in turn, via interaction with humoral factors, controls the fate in terms of tissue distribution of the carrier and its contents. Based on these studies, a rationale for liposome design aimed at in vivo drug delivery is discussed.
Lipid-conjugates of two amphipatic polymers, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ) (degree of polymerization approximately 50) were synthesized by linking glutarate esters of the polymers to distearoylphosphatidylethanolamine (DSPE) or alternatively by termination of the polymerization process with DSPE. Surface-modified liposomes (90 +/- 5 nm) prepared from either conjugate (5 mol % of total lipid) were injected into rats and followed by blood level and tissue distribution measurements. Both polymers PEOZ and PMOZ were found to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as polyethylene glycol (PEG), the best known material for this purpose. This is the first demonstration of protection from rapid recognition and clearance conveyed by alternative polymers, which is equal to the effect of PEG.
Newly synthesized amphiphilic polyacrylamide and poly(vinyl pyrrolidone), single terminus-modified with long-chain fatty acyl groups, are able to incorporate into the liposomal membrane, and similar to poly(ethylene glycol) prolong liposome circulation in vivo and decrease liposome accumulation in the liver. Protective efficacy of modified polymers increases with the increase in the length of acyl moiety and decreases for higher molecular weight polymers. The data on amphiphilic polymer-modified liposome biodistribution are presented.
The hypothetical model is built explaining the molecular mechanism of protective action of poly(ethylene glycol) on liposomes in vivo. The protective layer of the polymer on the liposome surface is considered as a statistical 'cloud' of polymer possible conformations in solution. Computer simulation was used to demonstrate that relatively a small number of liposome-grafted molecules of hydrophilic and flexible polymer can create a dense protective conformational cloud over the liposome surface preventing opsonizing protein molecules from contacting liposome. A more rigid polymer fails to form this dense protective cloud, even when hydrophilic. Computer simulation was also used to reveal possible heterogeneity of reactive sites on a polymer-coated liposome surface, and to estimate the optimal polymer-to-lipid ratio for efficient liposome protection. Experiments have been performed with the quenching of liposome-associated fluorescent label (nitrobenzoxadiazole or fluorescein) with protein (rhodamine-ovalbumin or anti-fluorescein antibody) from solution. It was shown that poly(ethylene glycol) grafting to liposomes hinders protein interaction with the liposome surface, whereas liposome-grafted dextran (more rigid polymer) in similar quantities does not affect protein-liposome interaction. Highly-reactive and low-reactive populations of chemically identical reactive sites have been found on polymer-coated liposomes. Experimental data satisfactory confirm the suggested mechanism for the longevity of polymer-modified liposome.
After intravenous (iv) injection of 125I-labeled poly(ethylene glycol) (PEG) with different molecular weights to mice, the radioactivity of the organs was measured to pharmacokinetically analyze the body distribution of PEG according to a two-compartment model. High molecular weight PEGs were retained in the blood circulation for a longer period than low molecular weight PEGs. The terminal half-life of PEG in the circulation extended from 18 min to 1 day as the PEG molecular weight increased from 6000 to 190,000. PEG tended to accumulate in the tissues/organs such as muscle, skin, bone, and the liver to a higher extent than the other organs, irrespective of the molecular weight. The time dependence of tissue accumulation was based on the vascular permeability. The results of pharmacokinetic analysis suggested that small PEG tended to freely translocate from the circulation to extravascular tissues and to return to the blood circulation again by diffusion, whereas large PEG translocated more slowly to extravascular tissues. Urinary clearance decreased with increasing PEG molecular weight, similar to the tissue clearance, whereas liver clearance increased with the increasing PEG molecular weight, after passing a minimum around the molecular weight of 50,000. PEG uptake by Kupffer cells was enhanced as the molecular weight became > 50,000.
Advances in therapeutic applications of liposomes have been achieved through surface modifications increasing their biological stability: reduced constituent exchange and leakage as well as reduced unwanted uptake by cells of the mononuclear phagocytic system. The recent conclusions obtained from in vivo and in vitro studies are reviewed with an emphasis on evaluating the methods used and thus the kinds of conclusions which can be drawn. A number of issues are raised as to the limitations of the methods employed. Steric stabilization, meaning reduction in particle interactions by a surface steric barrier, has been proposed as a theoretical basis for the results and some of the initial results testing this hypothesis are reviewed here with respect to identification of the extent to which physical properties of the surface coatings correlate with the biological properties. At this time it seems that no one method is ideal so that multiple measures give the best characterization.
Carboxy group-terminated synthetic polymers--branched poly(ethylene glycol), poly(acryloylmorpholine), and poly(vinylpyrrolidone)--were made amphiphilic by derivatization with phosphatidyl ethanolamine via the terminal carboxy group and then incorporated into lecithin-cholesterol liposomes prepared by the detergent dialysis method. Following the biodistribution of liposomes in mice, all three polymers were shown to be effective steric protectors for liposomes and were able to sharply increase liposome circulation times in a concentration-dependent manner. The accumulation of liposomes in the liver decreases. The effects observed are similar to those found for liposomes modified with linear poly(ethylene glycol). At low polymer concentration, amphiphilic branched poly(ethylene glycol) seems to be the most effective liposome protector, most probably, because at the same molar content of anchoring groups, each attachment point carries two polymeric chains and doubles the quantity of liposome-grafted polymer comparing to linear poly(ethylene glycol).
Biodistribution and infarct accumulation of different liposome preparations in rabbits with experimental myocardial infarction have been investigated. The influence of such parameters as liposome size, and presence or absence of poly(ethylene glycol) (PEG) and infarct-specific antimyosin antibody (AM) on liposome behavior in vivo was studied. All three variables were shown to affect liposome biodistribution, liposome size being the least significant variable. Statistical analysis of the data obtained demonstrated that of all variables, PEG coating expresses the strongest influence on the liposome blood clearance, significantly (P=0.0001) increasing the mean level of blood radioactivity under all circumstances. Infarct accumulation depended upon the presence of both PEG (P=0.0013) and AM (P=0.005). The infarct-to-normal ratio was affected by the presence of AM (P=0.0002), but the extent of the effect depended also on the presence of PEG (P=0.01). Two differing mechanisms can be seen in infarct accumulation of PEG-liposomes (slow accumulation via the impaired filtration) and AM-liposomes (specific binding of immunoliposomes with the exposed antigen). Both mechanisms are supplementary in case of liposomes carrying PEG and AM at the same time. An optimization strategy is suggested for using liposomes as carriers for diagnostic (a high target-to-nontarget ratio is required) and therapeutic (a high absolute accumulation in the target is required) agents.
There is an urgent need for more active and better tolerated chemotherapy regimens for the treatment of advanced breast and ovarian cancers. Current therapeutic strategies in these malignancies include the use of moderately effective initial regimens that are usually accepted by patients. Tolerability considerations are especially important in the development of palliative regimens: retreatment for persistent or hormone-resistant disease must include quality-of-life analyses.
Pegylated liposomal doxorubicin (PLD) has demonstrated a better therapeutic index than free doxorubicin in murine solid tumours and human tumour xenografts in nude mice. In early clinical studies in patients with refractory ovarian cancer, PLD has produced high response rates (26%) and gratifyingly long response durations (8 to 21 + months after onset of therapy). Less mature data also suggest that PLD is active against breast cancer.
Information from these same clinical studies confirms the marked reduction in several toxicities associated with free doxorubicin, including nausea and vomiting, myelosuppression and cardiotoxicity. Alopecia is also markedly diminished. On the other hand, mucosal and skin toxicities appear to be more common with PLD.
PLD therefore offers the prospect of retaining activity, together with attenuated acute toxicity. In addition to facilitating the development of palliative regimens with better tolerability, the drug may lend itself to effective integration of chemotherapy with loco-regional therapies, utilisation in ‘maintenance’ regimens that are associated with an acceptable quality of life for the patient, and the avoidance of long term toxicities associated with treatment. Moreover, additional study of PLD in combination with other drugs and modalities may extend the use of the drug beyond palliation to the development of combination regimens with other drugs at conventional doses, and high doses with G-CSF support.
The field of long-circulating microparticulate drug carriers is reviewed. The protective effect of certain polymers including poly(ethylene glycol) on nanoparticulate carriers (liposomes, nanoparticles, micelles) is considered in terms of statistical behaviour of macromolecules in solution. Using liposomes as an example, the mechanism is discussed assuming that surface-grafted chains of flexible and hydrophilic polymers form dense 'conformational clouds' preventing other macromolecules from interaction with the surface even at low concentrations of the protecting polymer. The scale of the protective effect is interpreted as the balance between the energy of the hydrophobic anchor interaction with the liposome membrane core or with the particle surface and the energy of the polymer chain free motion in solution. The possibility of using protecting polymers other than poly(ethylene glycol) is analysed, and examples of such polymers are given, based on polymer-coated liposome biodistribution data. General requirements for protecting polymers are formulated. Sterically protected nanoparticles and micelles are considered, and differences in steric protection of liposomes and particles are discussed. The problem of the preparation of drug carriers combining longevity and targetability is analysed. The biological consequences of steric protection of drug carriers with surface-grafted polymers are discussed, and possible clinical applications for long-circulating pharmaceutical carriers are considered.
To determine the efficacy and safety profile, including the risk for cardiac toxicity, of liposome-encapsulated doxorubicin (TLC D-99), fluorouracil (5-FU), and cyclophosphamide as first-line chemotherapy in patients with metastatic breast cancer (MBC).
Forty-one women were registered in this phase II study. All patients had measurable disease and no previous chemotherapy for MBC. Treatment consisted of TLC D-99 60 mg/m2 and cyclophosphamide 500 mg/m2 on day 1 and 5-FU 500 mg/m2 on days 1 and 8 every 3 weeks. Serial cardiac monitoring, including endomyocardial biopsies, was performed.
The overall response rate was 73% (95% confidence interval, 57% to 86%). The median duration of response was 11.2 months, the median time to treatment failure was 8.1 months, and the median overall survival duration was 19.4 months. The median number of cycles per patient was 10. The median cumulative dose of TLC D-99 was 528 mg/m2. Ten patients required hospitalization for febrile neutropenia. Nausea/vomiting, stomatitis, and fatigue higher than grade 2 occurred in 12%, 15%, and 41% of patients, respectively. Twenty-one patients reached a cumulative doxorubicin dose greater than 500 mg/m2. Three patients (7%) were withdrawn from the study due to protocol-defined cardiac toxicity, two because of a decrease in left ventricular ejection fraction to < or = 40%, and one because her endomyocardial biopsy result was grade 1.5. One patient had congestive heart failure that was probably nonanthracycline related.
This chemotherapy regimen, including TLC D-99, was highly active against MBC and associated with low cardiac toxicity despite high cumulative doses of doxorubicin.
For the purpose of providing a summary of current clinical trials to determine whether povidone-iodine is beneficial or detrimental to wound healing, an integrated review was completed. Clinical trials were defined as any study that uses some concentration and form of povidone-iodine in a comparison or evaluation with other products or treatments resulting in an impact of povidone-iodine on wounds. The use of povidone-iodine for cleansing, irrigating, and dressing wounds is controversial. Wound healing is complex and requires safe and effective treatment modalities. Numerous in vitro and in vivo studies have been done with conflicting results on bactericidal effects and cytotoxicity of this antimicrobial agent. Human and animal in vivo studies in the last 10 years were used for this review because often the relevance of in vitro data in clinical conditions are questioned. The varied studies provide evidence that in most instances, povidone-iodine did not effectively promote good wound healing; in fact, most studies showed either impaired wound healing, reduced wound strength, or infection.
Most solid tumors possess unique pathophysiological characteristics that are not observed in normal tissues or organs, such as extensive angiogenesis and hence hypervasculature, defective vascular architecture, impaired lymphatic drainage/recovery system, and greatly increased production of a number of permeability mediators. The phenomenon now known as the enhanced permeability and retention (EPR) effect for lipid and macromolecular agents has been observed to be universal in solid tumors. Primarily, enhanced vascular permeability will sustain an adequate supply of nutrients and oxygen for rapid tumor growth. The EPR effect also provides a great opportunity for more selective targeting of lipid- or polymer-conjugated anticancer drugs, such as SMANCS and PK-1, to the tumor. In the present review, the basic characteristics of the EPR effect, particularly the factors involved, are described, as well as its modulation for improving delivery of macromolecular drugs to the tumor. Tumor-specific vascular physiology is also described.
Polymers in solution. Their model-ling and structure
- J Cloizeaux
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des Cloizeaux J, Jannink G. Polymers in solution. Their model-ling and structure. Oxford: Clarendon Press, 1990.
Hand-book of water soluble gums and resins
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Powell GM. Polyethylene glycol. In: Davidson RL. editor. Hand-book of water soluble gums and resins. New York: McGraw-Hill, 1980, [Chapter 18].
Final report of the safety assessment of polyethylene glycols (PEGs)-6, -8, -32, -75, -150, -14M, -20M
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Pang SNJ. Final report of the safety assessment of polyethylene glycols (PEGs)-6, -8, -32, -75, -150, -14 M, -20 M. J Am Coll Toxicol 1993;12:429}56.
Why do polyethylene glycol-coated liposomes circulate so long?
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Torchilin VP, Papisov MI. Why do polyethylene glycol-coated liposomes circulate so long? J Liposome Res 1994;4:725}39.
Microparticulate systems for the delivery of proteins and vaccines
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Cohen S, Bernstein H, editors. Microparticulate systems for the delivery of proteins and vaccines. New York: Marcel Dekker, 1996.