Complex Formation Between The Anionic Polymer (PAA) and a Cationic Drug (Procaine HC1): Characterization by Microcalorimetric Studies

Purpose. Due to the importance of drug-polymer interactions in, inter alia, drug loading/release, supramolecular assemblies and DNA delivery for gene therapy, the aim of this study was therefore to establish the mechanism of interaction between a model polymer (Polyacrylic acid, PAA) and a model drug (procaine HCl). Methods. This was performed by studying the effect of salt (KCl) concentration on their heat released values using Isothermal Titration Microcalorimetry (ITM). The integrated released heat data were computer fitted to a one class binding model and the thermodynamic parameters (K_obs, H, and N) were determined. Results. As the KC1 concentration was increased, K_obs decreased thus establishing the salt dependence of the interaction. The linear variation of G_obs with S_obs indicated that their interaction was entropically driven. The stoichiometry of the interaction was calculated to be one procaine molecule per monomer of PAA. Dissection of the total observed free energy at each KC1 concentration indicated that the contribution of the non-electrostatic attractions to the interaction of PAA with procaine HC1 was greater than those of the electrostatic attractions. Conclusions. We have shown that the interaction between PAA and procaine HC1 is dependent upon the presence of counterions (monovalent ions) and is mainly entropically driven. The calculated stoichiometry indicated that one procaine HC1 molecule neutralised one carboxylic acid group on PAA. Although electrostatic interactions were necessary for initiating complex formation, the non-electrostatic forces were dominant in stabilising the PAA-procaine HC1 complex.     

Uptake of FITC-chitosan nanoparticles by A549 cells

Purpose. The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. Methods. Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. Results. Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having K_m and V_max of 3.84 M and 58.14 g/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K⁺ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37°C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37°C. Conclusions. Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.     

Physicochemical Parameters Responsible for the Affinity of Methotrexate Analogs for Rat Canalicular Multispecific Organic Anion Transporter (cMOAT/MRP2)

Purpose. Canalicular multispecific organic anion transporter (cMOAT/MRP2) is known to exhibit a broad substrate specificity toward amphiphatic organic anions, including methotrexate (MTX). The present study aims to identify the physicochemical properties of MTX derivatives that correlate with recognition specificity by cMOAT/MRP2. Methods. We examined the inhibitory effect of MTX and 24 analogs on the transport of [³H]–S–(2,4–dinitrophenyl)glutathione by cMOAT/MRP2. The affinity constants of these compounds were compared with their physicochemical parameters. The primary active transport of several compounds was also confirmed. Results. The affinity constants closely correlated with the octanol/water partition coefficient (clogP), and a linear combination of polar and nonpolar surface areas. The affinity for cMOAT/MRP2 also closely correlated with the molecular weight, which also showed a significant correlation with nonpolar surface area and clogP. Conclusions. Recognition by cMOAT/MRP2 depends on a balance of dynamic surface properties between the polar and nonpolar regions of MTX analogs. The so–called molecular weight threshold for the cMOAT/MRP2 affinity of these compounds can be explained by their physicochemical parameters, especially their nonpolar surface areas.     

Molecular Factors Influencing Retention on Immobilized Artificial Membranes (IAM) Compared to Partitioning in Liposomes and n-Octanol

Purpose. To assess the effect of molecular factors influencing retention on immobilized artificial membrane (IAM) high-performance liquid chromatography columns compared to liposomal partitioning and traditional n-octanol/water partition coefficients. Methods. IAM capacity factors were measured at pH 7.0 on an IAM.PC.DD2 stationary phase. Liposomal partitioning at pH 7.0 and n-octanol/water partition coefficients were measured using the pH metric method. Partitioning in egg-phosphatidylcholine (PhC) liposomes was also measured by equilibrium dialysis for a series of -blockers. Results. For the ionized -blockers, potentiometry and equilibrium dialysis yielded consistent partitioning data. For relatively large bases, IAM retention correlated well with PhC liposome partitioning, hydrophobic forces being mainly involved. For more hydrophilic compounds and for heterogeneous solutes, in contrast, the balance between electrostatic and hydrophobic interactions was not the same in the two systems. Hydrogen bonding, an important factor in liposomes partitioning, played only a minor role in IAM retention. Conclusions. Partitioning in immobilized artificial membranes depends on size, hydrophobicity, and charge. When hydrophobic interactions dominate retention, IAM capacity factors are well correlated with liposomal partitioning. On the contary, for hydrophilic solutes, the two systems do not yield the same information and are not interchangeable.     

Loading of Tetanus Toxoid to Biodegradable Nanoparticles from Branched Poly(Sulfobutyl-Polyvinyl Alcohol)-g-(Lactide-Co-Glycolide) Nanoparticles by Protein Adsorption: A Mechanistic Study

Purpose. Mucosal delivery of vaccine-loaded nanoparticles (NP) is an attractive proposition from an immunologic perspective. Although numerous NP preparation methods are known, sufficient antigen loading of NP remains a challenge. The aim of this study was to evaluate adsorptive loading of NP with a negatively charged surface structure using tetanus toxoid (TT) as a model vaccine. Methods. Blank NP, consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide), as well as poly(lactide-co-glycolide) NP were prepared by a solvent displacement technique. The use of polymers with different degrees of substitution resulted in NP with different negative surfaces charges. Adsorption of TT to NP was performed varying to NP surface properties, protein equilibrium concentration, and loading conditions. Results. The protein adsorption was controlled by NP surface properties, and maximum TT adsorption occurred at highly negatively charged NP surfaces. Results from isothermal titration calorimetry and -potential measurement suggest an adsorption process governed by electrostatic interactions. The adsorption followed the Langmuir isotherm in the concentration ranges studied. TT withstood this gentle loading procedure in a nonaggregated, enzyme-linked immunoabsorbant assay-active form. Conclusions. The results demonstrate that negatively charged NP consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide) are suitable for adsorptive loading with TT and may have potential for mucosal vaccination.     

Hydrogen Bonding and Electrostatic Interaction Contributions to the Interaction of a Cationic Drug with Polyaspartic Acid

Purpose. To determine the mechanism and identify forces of interactionbetween polyaspartic acid and diminazene (a model drug). Such knowledgeis essential for the design of polymeric drug delivery systemsthat are based on molecular self-assembly into complexes or micellartype systems. Methods. Complex formation was studied by isothermal titrationmicrocalorimetry and the McGhee von Hippel model was applied toobtain K _obs, H _obs, and n _obs. The calorimetry data were compared withboth an optical density study and the amount of free/complexed drug. Results. The diminazene-polyaspartic acid interaction is enthalpicallydriven, whereby one diminazene molecule interacts with two monomersof polyaspartic acid. The dependence of K _obs on saltconcentrationreveals a contribution of electrostatic interactions. However, applyingManning's counter ion condensation theory shows that the major drivingforce for the complex formation is hydrogen bonding, with interfacialwater molecules remaining buried within the complex. Themodelling of the pH dependence of K _obs and H _obsdemonstrates thatthe ionization of carboxylic groups of polyaspartic acid is a prerequisitefor the interaction. Conclusions. Complex formation between diminazene and polyasparticacid is driven by both electrostatic interactions and hydrogen bonding,with the latter being the dominating force. Although electrostaticinteractions are not the major driving force, ionization of the drug andpolymer is essential for complex formation.     

Study of the Complex Between the Contrast Agent lobitridol (Xenetix®) and Elastase (PPE): A Model for Hydrophobic Site Protection in Drug-Protein Interactions

Purpose. The concept of Hydrophilic Sphere Stabilization, or Hydrophobic Shielding, has been postulated in the synthesis of biocompatible contrast agents in vascular imaging. To improve the safety of these polyiodinated agents, interactions with protein hydrophobic sites in biomacromolecules should be kept as low as possible. In order to evaluate the level of interactions with proteins, we have selected the serine proteinase Elastase, in presence of Iobitridol (Xenetix^®), as a model. Methods. The complex between Iobitridol and Pancreatic Porcine Elastase was investigated by X-ray diffraction techniques, on saturated monocrystals, using the synchrotron radiation at 0.98. Results. In contrast to Iohexol, which displays several interactions including one in the active site, Iobitridol is unable to interact directly with elastase. Only one partially occupied site is found in between two molecules of the crystal packing. Conclusions. The validation of the 'hydrophobic shielding' concept, which was at the origin of the design of the Iobitridol molecule, has been proven to be an essential feature in minimizing in vivo protein interactions.     

Physical Properties of Solid Molecular Dispersions of Indomethacin with Poly(vinylpyrrolidone) and Poly(vinylpyrrolidone-co-vinyl-acetate) in Relation to Indomethacin Crystallization

Purpose. To measure solid-state features of amorphous molecular dispersions of indomethacin and various molecular weight grades of poly(vinylpyrrolidone), PVP, and poly(vinylpyrrolidone-co-vinylacetate), PVP/VA, in relation to isothermal crystallization of indomethacin at 30°C Methods. The glass transition temperatures (Tg) of molecular dispersions were measured using differential scanning calorimetry (DSC). FT-IR spectroscopy was used to investigate possible differences in interactions between indomethacin and polymer in the various dispersions. The enthalpy relaxation of 5% w/w and 30% w/w polymer dispersions was determined following various aging times. Quantitative isothermal crystallization studies were carried out with pure indomethacin and 5% w/w polymers in drug as physical mixtures and molecular dispersions. Results. All coprecipitated mixtures exhibited a single glass transition temperature. All polymers interacted with indomethacin in the solid state through hydrogen bonding and in the process eliminated the hydrogen bonding associated with the carboxylic acid dimers of indomethacin. Molecular mobility at 16.5°C below Tg was reduced relative to indomethacin alone, at the 5% w/w and 30% w/w polymer level. No crystallization of indomethacin at 30°C was observed in any of the 5% w/w polymer molecular dispersions over a period of 20 weeks. Indomethacin alone and in physical mixtures with various polymers completely crystallized to the form at this level within 2 weeks. Conclusions. The major basis for crystal inhibition of indomethacin at 30°C at the 5% w/w polymer level in molecular dispersions is not related to polymer molecular weight and to the glass transition temperature, and is more likely related to the ability to hydrogen bond with indomethacin and to inhibit the formation of carboxylic acid dimers that are required for nucleation and growth to the crystal form of indomethacin.     

Chemotherapeutic Drugs Released from Polymers: Distribution of 1,3-bis(2-chloroethyl)-l-nitrosourea in the Rat Brain

Purpose. The distribution of [³H]BCNU following release from polymer implants in the rat brain was measured and evaluated by using mathematical models. Methods. [³H]BCNU was loaded into p(CPP:SA) pellets, which were subsequently implanted intracerebrally in rats; [³H]BCNU was also directly injected into the brains of normal rats and rats with intracranially transplanted 9L gliomas. Concentrations of [³H]BCNU on coronal sections of the brain were measured by autoradiography and image processing. For comparison, the kinetics of [³H]BCNU release from the p(CPP:SA) polymer discs into phosphate-buffered saline were also measured. Results. High concentrations of BCNU (corresponding to ~1 mM) were measured near the polymer for the entire 30-day experiment. The penetration distance, defined as the distance from the polymer surface to the point where the concentration of [³H]BCNU in the tissue had dropped to 10% of the maximum value, was determined: penetration distance was ~5 mm at day 1 and ~1 mm at days 3 through 14. Local concentration profiles were compared with a mathematical model for estimation of the modulus ², an indicator of the relative rate of elimination to diffusion in the brain. From day 3 to 14, ² was ~7, indicating that BCNU elimination was rapid compared to the rate of diffusive penetration into tissue. The enhanced penetration observed on day 1 appears to be due to convection of extracellular fluid caused by transient, vasogenic edema, which disappears by day 3. Conclusions. Polymer implants produce very high levels of BCNU in the brain, but BCNU penetration into brain tissue is limited due to rapid elimination.     

Towards the Predictability of Drug-Lipid Membrane Interactions: The pH-Dependent Affinity of Propranolol to Phosphatidylinositol Containing Liposomes

Purpose. Prediction of the pH-dependent affinity of (RS)-[³H]propranolol to mixed phosphatidylcholine (PhC)/phosphatidylinositol(Phl) membranes from the partitioning in the single lipid liposome/buffer systems. Methods. Partition studies in liposome/buffer systems were performed by means of equilibrium dialysis at 37°C between pH 2 and 11 at a molar propranolol to lipid ratio of 10^–6 to 10^–5 in the membrane. Results. The Phl membrane more strongly attracts the protonated (RS)-[³H]propranolol than the neutral solute, i.e. the partition coefficient of the protonated base (P_i) is 17430 ± 1320, P of the neutral compound (P_n) is 3110 ± 1650. In the PhC-liposome system P_i is 580 ± 17, P_n 1860 ± 20. The partition coefficients show an exponential dependence on the molar Phl fraction in mixed liposomes. The partitioning in mixed PhC/Phl membranes is predictable from P_n and P_i in the single lipid liposome systems. Conclusions. The negative charge of biological lipid membranes causes strong electrostatic interactions with positively charged solutes. This strong attraction is not predictable from the octanol/buffer partition system, but it is important regarding drug accumulation in the tissue and drug attraction by certain lipids in the vicinity of membrane proteins.     

Transient Expression of a Purine-Selective Nucleoside Transporter (SPNTint) in a Human Cell Line (HeLa)

Purpose. The goal of this study was to develop a mammalian expression system for the cloned rat intestinal, Na⁺-dependent, purine-selective nucleoside transporter (SPNT_int) and to study the interactions of nucleosides and nucleoside analogs with this transporter. Methods. Lipofection was used to transfect HeLa cells with a mammalian expression vector (pcDNA3) containing the cDNA insert encoding SPNT_int. Nucleoside transport activity was measured using [³H] inosine, [³H]uridine, [³H]-dideoxyinosine (ddl), and [³H]-2-chloro-2-deoxyadenosine (2CdA) as model substrates. Results. Expression of SPNT_int was observed between 36 and 90 h post-transfection, with maximal expression at 66 h. At 66 h, Na⁺-stimulated uptake of [³H]inosine in cells transiently transfected with SPNT_int was approximately threefold greater than that in cells transfected with empty vector (p < 0.05). The Na⁺-stimulated uptake of both inosine and uridine was saturable (K_m = 28.1 ± 7.1 M and 20.6 ± 5.6 M, respectively) in the transfected cells and was significantly inhibited by the naturally occurring nucleosides (1 mM) inosine and uridine and to a lesser extent by thymidine. The nucleoside analogs ddl (IC₅₀ = 46 M) and 2CdA (IC₅₀ =.13 M) also significantly inhibited the Na⁺-stimulated uptake of [³H]inosine. A Na⁺-stimulated uptake of [³H]2CdA was observed suggesting that 2CdA is also a permeant of SPNT_int. Conclusions. HeLa cells transiently transfected with SPNT_int represent a useful tool to study the kinetics and interactions of drugs with SPNT_int.     

Synthesis of a Lipophilic Prodrug of 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) and Its Incorporation into a Hepatocyte-Specific Lipidic Carrier

Purpose. 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of Hepatitis B virus replication, is in vivo hardly taken up by parenchymal liver cells (the site of infection). Our aim is to examine whether lactosylated reconstituted HDL (LacNeoHDL), a lipidic particle that is specifically internalized by parenchymal liver cells, is a suitable carrier for the selective delivery of PMEA to this cell type. Methods. To incorporate PMEA into LacNeoHDL, we synthesized a lipophilic prodrug (PMEA-LO) by coupling PMEA via an acid-labile phosphonamidate bond to lithocholic acid-3-oleate. Results. The yield of the synthesis was 52% ([³H]PMEA-LO: 24%). [³H]PMEA-LO readily incorporated into LacNeoHDL (13 molecules/ particle) without affecting the size and net negative charge of the carrier. Further, incubation studies at lysosomal pH showed [³H]PMEA was completely released from the carrier whereas, at neutral pH or in plasma, appreciable release was not observed. Conclusions. The conjugation of PMEA with lithocholic acid-3-oleate results in a lipophilic prodrug that readily associates with LacNeoHDL. The association of the prodrug does not affect the physico-chemical properties of the particle, and PMEA is released from the carrier at lysosomal pH. These findings indicate that by using the prodrug approach, LacNeoHDL is a suitable carrier to deliver PMEA to parenchymal liver cells.     

Surface plasmon resonance-induced photoactivation of gold nanoparticles as mitochondria-targeted therapeutic agents for pancreatic cancer

Background: Noble metal nanoparticles such as gold nanoparticles can strongly absorb light in the visible region by inducing coherent collective oscillation of conduction band electrons in strong resonance with visible frequencies of light. This phenomenon is frequently termed as surface plasmon resonance (SPR).

Objectives: The main objective was to study the effects of laser photoactivated gold nanoparticles (by means of SPR) on human pancreatic cancer cells.

Results: Gold nanoparticles obtained using standard wet chemical methods (with sodium borohydride as a reducing agent) underwent photoexcitation using 2w 808 nm laser and further administered to 1.4E7 pancreatic cancer cell lines. Flow cytometry, transmission electron microscopy, phase contrast microscopy, quantitative proteomics and confocal microscopy combined with immunochemical staining were used to examine the interaction between photo excited gold nanoparticles and pancreatic cancer cells.

Conclusion: The study shows that phonon–phonon interactions following laser photoexcitation of gold nanoparticles exhibit increased intracellular uptake, as well as mitochondrial swelling, closely followed by mitochondrial inner membrane permeabilization and depolarization. This unique data may represent a major step in mitochondria-targeted anticancer therapies using laser-activated gold nanoparticles.     

Nonlinear Binding of Valproic Acid (VPA) and E-Δ2-Valproic Acid to Rat Plasma Proteins

The binding characteristics of valproic acid (VPA) and its pharmacologically active monounsaturated metabolite, E-²-VPA, to rat plasma proteins were compared. The plasma free fraction was determined by a rapid equilibrium procedure, which minimizes the interfering effects of nonesterified fatty acids liberated by in vitro lipolysis. Nonlinear binding behavior was observed with both compounds over their respective pharmacologic concentration range. Multiple binding-site models were invoked to explain the binding isotherm. The 2-unsaturated compound has a much higher affinity for the rat plasma proteins (mainly albumin) than its saturated precursor. The equilibrium association constants for the high- and intermediate-affinity sites were more than an order of magnitude higher with E-²-VPA than with VPA (10⁴–10⁶ versus 10³ M ^–1). This difference in binding affinity was also reflected by a lower plasma free fraction for E-²-VPA compared with VPA (<<10 versus >20% at total concentrations of less than 100 µg/ml). A more pronounced dose- and concentration-dependent variation in the distribution and clearance kinetics is predicted for the 2-unsaturated analogue compared to VPA. Also, the structural dependency in plasma protein binding observed with these branched-chain fatty acids may provide insights into the mechanism of interaction between fatty acyl molecules and albumin.     

Synthesis and In Vitro Evaluation of Chitosan-EDTA-Protease-Inhibitor Conjugates Which Might Be Useful in Oral Delivery of Peptides and Proteins

Purpose. To develop a novel mucoadhesive polymer that protects peptide drugs from degradation by secreted as well as membrane-bound proteases in the intestine, and to evaluate this polymer in vitro. Methods. The serine protease inhibitors antipain, chymostatin and elastatinal were covalently linked to chitosan (poly-[l 4]--D-glucosamine). Thereafter, the complexing agent ethylenediaminete-traacetic acid (EDTA) was bound to the remaining primary amino groups of the polymer. The inhibitory effect of the resulting polymer-conjugate towards trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) and aminopeptidase N (EC 3.4.11.2) as well as its mucoadhesive properties were evaluated in vitro. Results. Whereas the novel polymer-conjugate exhibited excellent swelling properties, its adhesive force was under our assay conditions 42% lower than that of unmodified chitosan. However, the polymer-conjugate showed a strong inhibitory activity towards all tested serine proteases. Due to its additional high binding affinity towards bivalent metal ions, it also inhibited the Zn²⁺-dependent exopeptidases carboxypeptidase A, B and aminopeptidase N. Conclusions. The novel mucoadhesive polymer-conjugate described in this study seems to be a useful tool in overcoming the enzymatic barrier to perorally administered therapeutic peptides and proteins.     

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins

Abstract

1.?This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR).

2.?Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (f_u?>?0.8) to extensive (f_u?<?0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound).

3.?The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP.

4.?Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.     

Conjugates of Antisense Oligonucleotides with the Tat and Antennapedia Cell-Penetrating Peptides: Effects on Cellular Uptake,Binding to Target Sequences,and Biologic Actions

Purpose. The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2-O-methyl phosphorothioate oligonucleotides. Methods. The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. Results. The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides. Conclusions. Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.     

A Solid-State NMR Study of Protein Hydration and Stability

Purpose. The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity. Methods. Magic angle spinning ¹³C, ¹⁵N, ¹H, ²H, and ¹⁷O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (₀, T₁) and low frequency (₁, T_1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays. Results. There was little change in T_1p observed with increasing humidity. The results show, however, that there is a decrease in T₁, for DNase, insulin and lysozyme at relative humidities ranging from 0–98%, and we propose that the reduction in T₁, is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using ¹⁷O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data. Conclusions. Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.     

Determination of Molecular Mobility of Lyophilized Bovine Serum Albumin and γ-Globulin by Solid-State 1H NMR and Relation to Aggregation-Susceptibility

Purpose. Feasibility of solid-state ¹H NMR for determining the mobility of protein molecules in lyophilized cakes was considered. The mobility in cakes with various levels of water content was studied in relation to aggregation-susceptibility. Methods. Spin-spin relaxation time (T₂) of protons in lyophilized bovine serum albumin (BSA) and -globulin (BGG) was measured as a function of hydration level by solid state ¹H NMR using a solid-echo pulse sequence. Moisture-induced aggregation of the lyophilized proteins was also determined by high performance size exclusion chromatography. Results. Lyophilized BSA and BGG became susceptive to aggregation when water content exceeded about 0.2 g/g of protein. T₂ of protein protons in the lyophilized cakes started to increase at lower water contents. The increase in aggregation susceptibility observed with increasing water content appears to follow the increase in T₂ of protein protons. For lyophilized BGG, both aggregation and T₂ of protein protons decreased at water contents above 0.5 g/g protein. Conclusions. Mobility of protein molecules in lyophilized cakes was successfully detemined by solid-state ¹H NMR. The aggregation susceptibility of proteins was strongly related to their molecular mobility as indicated by T₂.     

Interactions of nanoparticles with plasma proteins: implication on clearance and toxicity of drug delivery systems

Introduction: Intravenously injected nanoparticles, like any other foreign pathogen that enters the body, encounter multiple lines of defense intended to neutralize and eliminate the invading substance. Adsorption of plasma proteins on the nanoparticle surface is the first barrier of defense, which could lead to physical changes in the formulation, such as aggregation and charge neutralization, biochemical activation of defense cascades, and trigger elimination by multiple types of phagocytic cell.

Areas covered: In this review, recent knowledge on the mechanisms that govern the interactions of nanoparticles (micelles, liposomes, polymeric and inorganic nanoparticles) with plasma proteins is discussed. In particular, the role of the nanoparticle surface properties and protective polymer coating in these interactions is described. The mechanisms of protein adsorption on different nanoparticles are analyzed and the implications on the clearance, toxicity and efficacy of drug delivery are discussed. The review provides readers with the biological insight into the plasma/blood interactions of nanoparticles.

Expert opinion: The immune recognition of nanoparticles can seriously affect the drug delivery efficacy and toxicity. There is at present not enough knowledge on the mechanisms that dictate the nanoparticle immune recognition and stability in the biological milieu. Understanding the mechanisms of recognition will become an important part of nanoparticle design.