"SMART" drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers.

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG(2000)-PE or PEG(5000)-PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 min) at lower pH values (pH 5.0-6.0), nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.

ATP-loaded liposomes effectively protect mechanical functions of the myocardium from global ischemia in an isolated rat heart model.

ATP-loaded liposomes (ATP-L) infused into Langendorff-instrumented isolated rat hearts protect the mechanical functions of the myocardium during ischemia/reperfusion. The left ventricular developed pressure (LVDP) at the end of the reperfusion in the ATP-L group recovered to 72% of the baseline (preservation of the systolic function) compared to 26%, 40%, and 51% in the groups treated with Krebs-Henseleit (KH) buffer, empty liposomes (EL), and free ATP (F-ATP), respectively. The ATP-L-treated group also showed a significantly lower left ventricular end diastolic pressure (LVEDP; better preservation of the diastolic function) after ischemia/reperfusion than controls. After incubating the F-ATP and ATP-L with ATPase, the protective effect of the F-ATP was completely eliminated because of ATP degradation, while the protective effect of the ATP-L remained unchanged. Fluorescence microscopy confirmed the accumulation of liposomes in ischemic areas, and the net ATP in the ischemic heart increased with ATP-L. Our results suggest that ATP-L can effectively protect myocardium from ischemic/reperfusion damage.

Encapsulation of ATP into liposomes by different methods: optimization of the procedure.

Different methods and conditions for ATP incorporation into PEGylated liposomes were compared in order to obtain a preparation with a maximized ATP content. Such a preparation may find the application for the in vivo treatment of ischemic tissues suffering from an insufficient ATP supply. Several different methods of liposome preparation and purification were used and HPLC was employed to determine the concentration of ATP in the liposomes. Thin lipid film hydration produced vesicles with the lowest ATP encapsulation (ca. 5 mol%). A pH gradient method yielded liposomes with ca. 10 mol% of ATP. Reverse phase evaporation and freezing-thawing methods resulted in a maximum entrapment of ATP on the level of 36-38 mol%. The freezing-thawing method was chosen for further investigation because of its simplicity and absence of a need to use organic solvents. The separation of the non-entrapped ATP by gel-filtration, centrifugation or dialysis yielded virtually identical liposomal preparations. The incorporation of PEG (as PEG-distearoyl phosphatidylethanolamine, PEG-DSPE) into the liposomal membrane decreases the quantity of the entrapped ATP (from 38 mol% for liposomes with 0.5 mol% of PEG-DSPE to only 17 mol% for liposomes with 5 mol% of PEG-DSPE).

TAT-liposomes: a novel intracellular drug carrier.

TAT peptide was attached to the surface of plain and PEGylated liposomes. These TAT peptide-modified liposomes have been shown to translocate into a variety of normal and cancer cells if a non-hindered interaction between the cell surface and liposome-attached TAT peptide was made possible. TAT peptide-liposomes translocated into cells remain intact within first few hours as proved by a co-localization of fluorescent markers entrapped inside liposomes and incorporated into the liposomal membrane. After 2 hours liposomes had slowly migrating towards cell nuclei. Liposomes had completely disintegrated with their inner marker released by approximately 9 hours. TAT peptide-liposomes were made slightly cationic by adding up to 10 mol %. of a cationic lipid (DOTAP). These slightly cationic liposomes were non-toxic towards cells, formed firm complexes with DNA (plasmid encoding for the formation of the Green Fluorescent Protein), and efficiently transfected a variety of cells. TAT peptide-liposomes can be considered as promising carriers for the non-endocytotic intracellular delivery of drugs and DNA.

Amphiphilic poly-N-vinylpyrrolidones: synthesis, properties and liposome surface modification.

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 micromolar 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. 10 mol% 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 1,500 and 8,000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors.

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide x cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Negatively charged polymers protect antinuclear antibody against inactivation by acylating agents.

For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, (111)In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives.

p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups.

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.

The putative role of phospholamban in the regulation of the heart muscle.

We describe the 50fold purification of phospholamban, the production of antibodies against it and preliminary experiments of reconstitution of purified phospholamban into vesicles of skeletal sarcoplasmic reticulum (SR). Purified phospholamban migrates in a modified Laemmli SDS polyacrylamide gel electrophoresis (PAGE) system with a relative molecular mass (Mr) of 22 kD and 6 kD. The higher Mr form is detectable by immunoreaction on Western blots only in heart SR preparations, whereas the low Mr form is also present in sarcolemmal preparations. No immunoreactivity was found in SR of skeletal muscle. Reconstituted skeletal SR vesicles were not influenced by phospholamban in respect to their Ca++-ATPase activity and calcium accumulation rate, but the obtained data suggest that phospholamban alters the calcium storage capacity of SR.

[Fractionation of fragments of skeletal muscle sarcoplasmic reticulum according to their sensitivity to caffeine]. / Fraktsionirovanie fragmentov sarkoplazmaticheskogo retikuluma skeletnoi myshtsy po ikh chuvstvitel'nosti k kofeinu.

Sarcoplasmic reticulum fragments were fractionated according to the ability of caffeine to selectively block Ca2+ uptake in the population of caffeine-sensitive membranes. The membrane suspension was loaded with calcium in the presence of oxalate, Mg-ATP and caffeine, after which the Ca2+-loaded caffeine-sensitive fragments were separated by sucrose density gradient centrifugation. In Ca2+-unloaded fragments of the supernatant, the sensitivity to caffeine estimated by its ability to diminish the rate of Ca2+ uptake, Ca/ATP ratio and Ca-oxalate capacity amounted to 91-93%. The terms of protein composition, the caffeine-sensitive fragments were identified with terminal cystern membranes, while the caffeine-insensitive ones with the SR canalicular membranes. The sensitivity to caffeine may serve as a reliable criterion for estimating the relative content of terminal cystern fragments in different microsomal preparations.

Ca2+ binding and charge movements in membranes of platelets and sarcoplasmic reticulum.

The properties of the Ca2+-pump system of platelet microsomes isolated without Ca2+-precipitating anions are studied. Passive Ca2+ binding to the microsomes takes place in a noncooperative manner with Kd = 0.7 microM. Half-maximal stimulation of ATP-dependent transport occurs at 0.4 microM Ca2+. The velocity of Ca2+ uptake, Ca2+ capacity and the level of phosphoprotein in platelet microsomes are significantly lower than in cardiac microsomes. Energization of platelet and muscle microsomes and activation of intact platelets result in opposite charge redistribution in hydrophobic regions of the membranes. It is concluded that these charge movements are caused by Ca2+ binding to and dissociation from nonpolar binding sites in the membranes.

The cardiac relaxing system. Its nature, calcium ion capacity, and influence of hydrogen and magnesium ions on initial velocity of calcium binding.

The functional and structural properties of Ca2+ ATPases isolated from heart and skeletal muscles were compared. The pH and Ca2+ dependences of the activities as well as amino acid and phospholipid composition of the enzymes are similar. On the other hand, specific activities of Ca2+ ATPases and their abilities to pump calcium in the reconstituted proteoliposomes differ. The sarcoplasmic reticulum (SR) extracted completely from 1 g of pigeon and guinea pig hearts is able to bind up to 65 and 76 nmol Ca2+/sec, respectively, at 37 degrees (24 microM concentration of free Ca2+ ions). In the absence of oxalate, the process of calcium binding is nonlinear in a time interval of 100 msec to 5 min. One-third and half of the quantity of calcium consumed at 1 sec is bound at 100 and 200 msec, respectively. Judging by steady-state levels of the phosphorylated intermediate of Ca2+ ATPase in highly purified and completely extracted preparations of SR, an estimate was made that 1 g of heart contains from 2 to 3 mg of SR protein. The rate of energy-dependent calcium binding by isolated cardiac SR depends on pH and the concentrations of free calcium and magnesium. At calcium concentrations above 5 microM, the rate of calcium accumulation is higher at pH 6.2 than at pH 7.2. Increase in magnesium ion concentration from 0.5 to 6.0 mM leads to a significant inhibition of calcium binding at calcium concentrations 0.1 to 10 microM. The data obtained show that at physiological concentrations of calcium, the ability of SR to accumulate calcium is close to that postulated for a system of calcium transport providing relaxation for heart muscle.

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum.

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopycnic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg [gamma-32P]ATP significant amounts of 32P were incorporated into all these proteins. Incorporation of 32P into the 15 000 dalton protein was moderately and 32P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by Ca2+ concentrations up to 0.1 mM and by ethyleneglycol-bis-(beta-aminoethyl-ether)-N,N'-tetraacetic acid in the absence of added Ca2+. Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [32P]-phosphate were incorporated into threonine and serine residues of this protein, when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP. The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.

The role of creatine phosphokinase in supplying energy for the calcium pump system of heart sarcoplasmic reticulum.

An investigation of isolated and purified heart sarcoplasmic reticulum performed in the current study indicates the presence of significant creatine phosphokinase (CPK) activity in this preparation. The localization of CPK on the membrane of sarcoplasmic reticulum has been revealed also by an electron microscopic histochemical method. Under the conditions of the Ca(2+)-ATPase reaction in the presence of creatine phosphate, the release of creatine into the reaction medium is observed, the rate of the latter process being dependent on the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the reticular membrane is able to maintain the high rate of calcium consumption by the sarcoplasmic reticulum vesicles. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of sarcoplasmic reticulum and indicate the important functional role of CPK in supplying energy for the Ca(2+)-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum.

[The functional coupling between Ca2+-ATPase and creatine phosphokinase in heart muscle sarcoplasmic reticulum]. / Funktsional'noe sopriazhenie mezhdu Ca2+-ATPazoi i kreatinfosfokinazoi v sarkoplazmaticheskom retikulume serdechnoi myshtsy.

The functional role of creatine phosphokinase (CPK) in the process of energy supply for the Ca2+-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum (SR) has been studied. It has been shown that isolated and purified preparations of heart SR contain significant activity of CPK. The localization of CPK on the membrane of SR has been revealed also by an electron microscopic histochemical method. Under conditions of the Ca+-ATPase reaction in the presence of creatine phosphate the release of creatine into the reaction medium is observed, the rate of the latter process being dependent upon the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the SR membrane is able to maintain higher rate of calcium uptake by SR vesicles, as compared to that with added ATP-regenerating system. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of SR.

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle.

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.

[Isolation of highly active preparations of sarcoplasmic reticulum and Ca2-dependent ATPase from cardiac muscle]. / Vydelenie vysokoaktivnykh preparatov sarkoplazmaticheskogo reticulum i Ca2-zavisimoi atpazy iz serdechnoi myshty

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.