Intracellular delivery of VIP-grafted sterically stabilized phospholipid mixed nanomicelles in human breast cancer cells

The purpose of this study was to determine whether biocompatible and biodegradable vasoactive intestinal peptide-grafted sterically stabilized phospholipid mixed nanomicelles (VIP-SSMM; size, approximately 15 nm), a novel nanosized actively targeted drug delivery platform for breast cancer, accumulate in human MCF-7 breast cancer cells. Using hydrophobic CdSe/ZnS quantum dots (QD), we found that QD-loaded VIP-SSMM accumulated significantly faster and in greater quantity in MCF-7 cells than did QD-loaded SSMM alone (p<0.05). This process was mediated, in part, by VIP receptors because excess human VIP, but not PACAP(6-38) or galanin, significantly attenuated this response (p<0.05). Taken together, these data indicate that VIP-SSMM are actively targeted to human breast cancer cells through VIP receptors. We suggest that VIP-SSMM could be used as an actively targeted nanosized drug delivery platform for breast cancer cells over-expressing VIP receptors.     

A lyophilized sterically stabilized liposome-containing docetaxel: in vitro and in vivo evaluation

Objectives: In this study, an improved lyophilized PEGylated liposomal formulation of docetaxel (DOC) has been developed.

Methods: PEGylated docetaxel liposome (PL-DOC) was prepared by thin-film evaporation method and lyophilization. The effect of various components of the lipids and their compatibility with DOC on the entrapment efficiency (EE) of liposome was investigated. The lyophilized PL-DOC was characterized by morphology, particle size, zeta potential, EE, release in vitro and stability. Pharmacokinetics and biodistribution in vivo of lyophilized PL-DOC were also investigated.

Results: The optimal liposome formulation was egg phosphatidylcholine (EPC):cholesterol (CH):DSPE-PEG2000:DOC?=?56:40:4:4 (molar ratio). Sucrose and mannitol were chosen as cryoprotectant in the lyophilization (cryoprotectant-to-lipid (C/L) mass ratio = 8:1). The size of lyophilized PL-DOC was 152.3?±?1.0?nm with negative charge and the EE was 89.75?±?1.79%. Compared with nonlyophilized PL-DOC, the lyophilized PL-DOC was more stable at 4?°C for six months. The lyophilized PL-DOC also showed the good stability after reconstituted by 5% glucose injection. In vitro release study of PL-DOC showed that PL-DOC had a sustained release effect. After i.v. administration at the dose of 10?mg/kg in rats, a significant increase in the AUC_0-∞, MRT_0-∞ and t_1/2 was observed in PL-DOC group compared with conventional docetaxel liposome (CL-DOC) and DOC injection (DOC-I) group. Biodistribution studies in mice showed that PL-DOC significantly decreased the uptake by the organs of mononuclear phagocytic system (MPS), such as liver and spleen, while prolonging the retention time of DOC in the plasma.

Conclusion: Our PEGylated liposome formulation reported in this study could potentially produce viable clinical strategies for improved delivery of DOC for the treatment of human cancer.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

Kinetics of stearic acid transfer between human serum albumin and sterically stabilized liposomes

The kinetics of the transfer of stearic acids between human serum albumin (HSA) and long circulating sterically stabilised liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) and of submicellar content of the polymer-lipid poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) have been studied by fluorescence spectroscopy. The study exploits the fact that HSA has a single tryptophan (Trp) residue and that the intrinsic Trp-emission intensity is quenched by the presence of doxyl spin-labelled stearic acids (SASL). Protein/lipid dispersions are considered in which SASL molecules are inserted either in the protein or in the SSL, and the transfer of SASL between the protein and SSL is conveniently monitored by the time variation of the inherent Trp-fluorescence intensity of HSA. It was found that the transfer of fatty acids between HSA and SSL depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers) and on the grafting density of the PEG-lipids at the lipid/protein interface. In the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer, and it is higher for the passage from DPPC bilayers to HSA. The presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer chains, especially at low grafting density and for lipid membranes in the fluid phase.     

Interactions of cytochromec with phospholipid membranes

Summary Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015m KCl is readily bound. After successive washings with 0.15m KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 170, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10m. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.     

Interactions of phospholipid monolayers with carbohydrates

Surface pressure studies of phospholipid monomolecular films of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) formed at an air/water interface have been made and the effects on the films studied when various carbohydrates are present in the subphase. The results obtained show that at a given temperature, the area per molecule of DPPC increases with increasing concentration of the carbohydrate in the subphase. The carbohydrate which has the greatest expanding effect on the phospholipid monolayer is glycerol, followed in turn by trehalose, sucrose, glucose, raffinose, and inositol. The mechanism of monolayer expansion by glycerol is different from that observed in other carbohydrates, as the following experiments demonstrate. Below the phase transition temperature of DPPC, the area per molecule of DPPC at a pressure of 12.5 dyn/cm is the same with and without glycerol in the subphase. However, when the monolayer is heated to a temperature above the phase transition temperature for DPPC, the area/molecule on glycerol is considerably greater than the area/molecule on water at the same surface pressure. Cooling the monolayer back to the lower temperature produces an area/molecule of DPPC which is identical on both water and glycerol subphases. Glycerol therefore has no effect on the low-temperature (condensed) monolayers but causes expansion of the high-temperature (expanded) monolayers. By contrast with glycerol, both trehalose and sucrose interact with the DPPC monolayer producing an increased area/molecule over that observed on water, both with low-temperature (condensed) monolayers and with the high-temperature (expanded) monolayers. The efficiency of these carbohydrates at expanding the monolayer films (with the exception of glycerol) shows a strong correlation with their ability to stabilize membrane structure and function at low water contents.     

Interactions of lipid micelles with blood proteins

Interaction of lipid micelles (LM), containing cholesterol and hydroxycholesterol, with human serum lipoproteins was investigated. It was shown that cholesterol-containing LM interact with low density lipoproteins (LDL). Selectivity of LM-LDL interaction depended on the cholesterol content of micelles and almost did not depend on the composition of LM core. Up to 90% of LDL were bound with cholesterol-saturated LM. By means of gel chromatography it was shown that interaction of cholesterol- and 7-hydroxycholesterol-containing micelles with serum led to the partial fusion of LDL with LM and LDL-LM complex formation, as well as to the cholesterol and 7-hydroxycholesterol transfer from micelles to LDL. The obtained results indicate that cholesterol-containing LM can be used for the delivery of oxidized cholesterol to cells involving LDL and receptor-dependent pathway of their capture by peripheral cells.     

Formyl peptide-induced contraction of human airways in vitro

Formylmethionylleucylphenylalanine (FMLP) is a synthetic analogue of bacterial chemotactic factors. We studied the contraction of human airway tissue in vitro by FMLP. FMLP induced a concentration-dependent contraction of all bronchial spiral strips studied (n = 45). The maximum tension generated in response to FMLP was 86.6 +/- 7.0% (SE) of the maximum response to histamine. The contraction was not reduced by the histamine H1-receptor antagonist pyrilamine, the cyclooxygenase and lipoxygenase inhibitors indomethacin and BW755C, the muscarinic antagonist atropine, or capsaicin which depletes stores of substance P. The concentration-response curve was shifted to the right by the polypeptide antagonist N-t-BOC-phenylalanylleucylphenylalanylleucylphenylalanine and the leukotriene antagonist FPL 55712. When 2 successive FMLP concentration-response curves were performed the maximum response was significantly reduced from 114.8 +/- 9.1% of the histamine maximum to 39.3 +/- 6.1%. The contraction of human airways in vitro by an agent that is structurally and functionally similar to chemotactic peptides released from bacteria may have important implications in airway disease.     

A growth factor antagonist as a targeting agent for sterically stabilized liposomes in human small cell lung cancer

The ability of a growth factor antagonist, [D-Arg(6),D-Trp(7,9)-N(me)Phe(8)]-substance P(6-11), named antagonist G, to selectively target polyethylene glycol-grafted liposomes (known as sterically stabilized liposomes) to a human classical small cell lung cancer (SCLC) cell line, H69, was examined. Our results showed that radiolabeled antagonist G-targeted sterically stabilized liposomes (SLG) bound to H69 cells with higher avidity than free antagonist G and were internalized (reaching a maximum of 13000 SLG/cell), mainly through a receptor-mediated process, likely involving clathrin-coated pits. This interaction was confirmed by confocal microscopy to be peptide- and cell-specific. Moreover, it was shown that SLG significantly improved the nuclear delivery of encapsulated doxorubicin to the target cells, increasing the cytotoxic activity of the drug over non-targeted liposomes. In mice, [(125)I]tyraminylinulin-containing SLG were long circulating, with a half-life of 13 h. Use of peptides like antagonist G to promote binding and internalization of sterically stabilized liposomes, with their accompanying drug loads, i.e., anticancer drugs, genes or antisense oligonucleotides, into target cells has the potential to improve therapy of SCLC.     

Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy

CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly ¹⁵N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q = 0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the receptor-containing bilayers immobilizes IL-8 (on the ∼ 10⁵ Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.     

Fluorine-18-labeled phospholipid quantum dot micelles for in vivo multimodal imaging from whole body to cellular scales

We have designed new nanoprobes applicable for both positron emission tomography (PET) and optical fluorescence in vivo imaging. Fluorine-18, which is commonly used for clinical imaging, has been coupled to phospholipid quantum dot (QD) micelles. This probe was injected in mice and we demonstrated that its dynamic quantitative whole body biodistribution and pharmacokinetics could be monitored using PET as well as the kinetics of their cellular uptake using in vivo fibered confocal fluorescence imaging. Phospholipid micelle encapsulation of QDs provides a highly versatile surface chemistry to conjugate multiple chemicals and biomolecules with controlled QD:molecule valency. Here, we show that, in contrast with several previous studies using other QD polymer coatings, these phospholipid QD micelles exhibit long circulation half-time in the bloodstream (on the order of 2 h) and slow uptake by reticulo-endothelial system.     

Interactions of cytochromes b5 and c with phospholipid monolayers

Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density.     

Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles

A peptide corresponding to the BH3 region of the proapoptotic protein, BID, could be bound in the cleft of the antiapoptotic protein, BCL-w. This binding induced major conformational rearrangements in both the peptide and protein components of the complex and led to the displacement and unfolding of the BCL-w C-terminal alpha-helix. The structure of BCL-w with a bound BID-BH3 peptide was determined using NMR spectroscopy and molecular docking. These studies confirmed that a region of 16 residues of the BID-BH3 peptide is responsible for its strong binding to BCL-w and BCL-x(L). The interactions of BCL-w and the BID-BH3 peptide complex with dodecylphosphocholine micelles were characterized and showed that the conformational change of BCL-w upon lipid binding occurred at the same time as the release and unfolding of the BH3 peptide.     

Protein refolding in reversed micelles: Interactions of the protein with micelle components

A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, gamma-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since gamma-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.     

Interactions of VIP with rigid phospholipid bilayers: implications for vasoreactivity

The purpose of this study was to determine whether vasoactive intestinal peptide (VIP), a pleiotropic amphipathic peptide, interacts with rigid liposomes composed of gel phase phospholipids. We found that incubation of VIP with small unilamellar gel phase liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and egg phosphatidylglycerol (ePG) for 2h at room temperature had no significant effects on VIP secondary structure. Moreover, suffusion of VIP (0.01, 0.1 and 1.0nmol) incubated in saline or with DPPC/ePG liposomes (size, 30 and 100nm) for 2h at room temperature or 4 degrees C onto the intact hamster cheek pouch microcirculation elicited a similar concentration-dependent vasodilation except for 0.01nmol VIP (P<0.05). By contrast, incubation of VIP with gel phase liposomes overnight at 4 degrees C significantly potentiated vasodilation evoked by all three concentrations of the peptide in comparison to aqueous VIP (P<0.05). VIP-induced vasodilation was liposome size-independent. The ratio of VIP to phospholipids in DPPC/ePG liposomes was concentration-independent. Collectively, these data indicate that short-term interactions of VIP with rigid phospholipid bilayers are limited resulting in only modest effects on VIP vasoreactivity in vivo.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.     

Synthesis, encapsulation, purification and coupling of single quantum dots in phospholipid micelles for their use in cellular and in vivo imaging

A detailed protocol for the synthesis of core/shell semiconductor nanocrystal, their encapsulation into phospholipid micelles, their purification and their coupling to a controlled number of small molecules is given. The protocol for the core/shell quantum dot (QD) CdSe/CdZnS synthesis has been specifically designed with two constraints in mind: green and reproducible core/shell QD synthesis with thick shell structure and QDs that can easily be encapsulated in poly(ethylene glycol)-phospholipid micelles with one QD per micelle. We present two procedures for the QD purification that are suitable for the use of QD micelles for in vivo imaging: ultracentrifugation and size-exclusion chromatography. We also discuss the different coupling chemistry for covalently linking a controlled number of molecules to the QD micelles. The total time durations for the different protocols are as follows: QD synthesis: 6 h; encapsulation: 15 min; purification: 1-4 h; coupling: reaction dependent.     

Interactions of human cytomegalovirus with human fibroblasts

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Interactions of human cytomegalovirus with human fibroblasts. J. Bacteriol. 91:213-220. 1966.-Virus attachment of human cytomegalovirus to human embryo lung fibroblasts was found to be temperature-independent, from 4 to 37 C. Prolonged incubation at 4 C, however, resulted in inactivation of a high proportion of attached virus. Virus penetration seemed to be temperature-dependent, occurring at 37 C but not at 4 C. Detailed studies of the growth curve of the virus were made. Cell-associated virus preceded the appearance of virus in the fluid phase by 2 to 5 days. Complement-fixing antigen could be detected, but only when the cytopathic effect was advanced, and it was demonstrable only in the cell-associated fraction. Under methyl cellulose, decreasing the bicarbonate concentration in the overlay from 0.225 to 0.15% resulted in marked increase in plating efficiency with all strains tested. However, varying the concentration of bicarbonate from 0.3 to 0.15% in fluid medium did not influence the growth of virus.