Melvin E. Klegerman

In vivo targeting of acoustically reflective liposomes for intravascular and transvascular ultrasonic enhancement.

OBJECTIVESnThe purpose of this study was to target acoustically reflective liposomes to atherosclerotic plaques in vivo for ultrasound image enhancement.nnnBACKGROUNDnWe have previously demonstrated the development of acoustically reflective liposomes that can be conjugated for site-specific acoustic enhancement. This study evaluates the ability of liposomes coupled to antibodies specific for different components of atherosclerotic plaques and thrombi to target and enhance ultrasonic images in vivo.nnnMETHODSnLiposomes were prepared with phospholipids and cholesterol using a dehydration/ rehydration method. Antibodies were thiolated for liposome conjugation with N-succinimidyl 3-(2-pyridyldithio) propionate resulting in a thioether linkage between the protein and the phospholipid. Liposomes were conjugated to antifibrinogen or anti-intercellular adhesion molecule-1 (anti-ICAM-1). In a Yucatan miniswine model, atherosclerosis was developed by crush injury of one carotid and one femoral artery and ingestion of a hypercholesterolemic diet. After full plaque development the arteries were imaged (20-MHz intravascular ultrasound catheter and 7.5-MHz transvascular linear probe) after injection of saline, unconjugated liposomes and antibody conjugated liposomes.nnnRESULTSnConjugated liposomes retained their acoustically reflective properties and provided ultrasonic image enhancement of their targeted structures. Liposomes conjugated to antifibrinogen attached to thrombi and fibrous portions of the atheroma, whereas liposomes conjugated to anti-ICAM-1 attached to early atheroma.nnnCONCLUSIONSnOur data demonstrate that this novel acoustic agent can provide varying targeting with different antibodies with retention of intravascular and transvascular acoustic properties.

In Vitro Targeting of Acoustically Reflective Immunoliposomes to Fibrin Under Various Flow Conditions

We have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b). The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Flow conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2 (15 dynes/cm2) over 70% of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.

Interaction Between Fibronectin‐bearing Surfaces and Bacillus Calmette‐Guérin (BCG) or Gelatin Microparticles

Gelatin, prepared commercially by degradation of animal collagen, was studied to see whether it had an affinity for fibronectin, which has a known affinity for collagen, and whether gelatin‐based drugs could be used to target fibronectin‐excreting tumours.

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.

Inhibition of murine sarcoma cell adherence to polystyrene substrata by bacillus Calmette-Guérin: evidence for fibronectin-mediated direct antitumor activity of BCG.

Bacillus Calmette-Guérin (BCG) inhibited adherence of S180 mouse sarcoma cells and WI38 human diploid fibroblasts to the polystyrene substratum of 24-well cluster dishes in a dose-dependent manner. This property was retained by washed or heat-killed bacilli, but not by the vaccine filtrate or by the spent bacterial culture medium. Adhesion of bacilli to nonadherent S180 cells was demonstrated by light and scanning electron microscopy, but was not seen after trypsinization of adherent cells, indicating that bacilli bind to cell-surface adhesins. Preincubation of bacilli with human fibronectin abolished their ability to inhibit S180 adherence, suggesting that the phenomenon may be mediated by interaction of bacilli with cell-surface fibronectin. Fibronectin pretreatment of the bacteria also decreased their inhibition of S180 tumor growth in vivo, indicating that this mechanism may be at least partly responsible for BCG vaccines observed antineoplastic activity.

A scanning electron microscope study of mycobacterial developmental stages in commercial BCG vaccines

Scanning electron microscopy (SEM) studies were performed on freshly prepared and freeze-dried Tice™-substrainMycobacterium bovis BCG vaccine as well as Tice BCG grown on Middlebrook 7H10 agar. Intact colonies of the Tice and Glaxo BCG substrains growing on agar were also examined. The presence of developmental stages of the mycobacterial life cycle previously reported in the literature was confirmed in actively growing BCG and in commercial vaccine preparations. The pleomorphic forms consisted of various size coccal and bacillary cells. Propagation appeared to occur by fission of both forms to produce aggregate bodies and by a coccal-bacillary cycle. Filterable (30–200 nm) granular cocci and coccal microcolonies were also observed in commercially prepared BCG vaccines. The implications of pleomorphism on the biologic activities of various BCG vaccines are discussed.

In‐vivo and In‐vitro Targeting of a Murine Sarcoma by Gelatin Microparticles Loaded with a Glycan (PS1)

Abstract— PS1, a complex polysaccharide derived from Mycobacterium bovis (Bacillus Calmette‐Guérin, BCG) with considerable antitumour activity in‐vivo, was loaded onto gelatin microparticles (mean diam. 1·45 μm) at a level shown to not produce the burst effect often seen with drug‐loaded micro‐particulate systems. In‐vitro dissolution experiments had demonstrated a sustained‐release behaviour, with a half‐life of approximately 8 h for what is an extremely water‐soluble material. These PS1/gelatin systems had no measurable cytotoxicity against an S180 murine sarcoma cell in‐vitro although fibronectin‐mediated targeting of the microparticles for the tumour cells could be demonstrated. Injection into mice, with the S180 cells, of PS1 solutions or suspensions of PS1‐loaded gelatin microparticles resulted in almost identical dose‐related suppression for the tumour cell growth. When injected at intervals following injection of the tumour cells, however, for a period of 24–48 h there was a relatively enhanced activity of the formulated PS1, compared with the aqueous solution, after which both formulated and unformulated material became progressively less effective.

Analysis of antineoplastic polysaccharides from Mycobacterium bovis BCG vaccine by high performance anion exchange chromatography with pulsed amperometric detection

Abstract Aqueous extracts of Mycobacterium bovis BCG vaccine, Tice® substrain, showed potent antineoplastic activity against a murine S180 sarcoma cell line in vivo. Following Sephadex LH-20 chromatography, one fraction (PSIA) was found to have antineoplastic activity, and contains complex polysaccharides. PS1A was further separated into four subfractions by Sephadex G-75 column chromatography. An HPLC method has been established for the analyses of polysaccharides in PS1A and its subfractions. Using a Dionex DX500 HPLC system, separation was achieved on a CarboPac PA1 anion exchange column by gradient elution with 0.1 M NaOH and 0.9 M NaOH/1 M NaOAc. Pulsed amperometry with three potential waveforms (E1 =0.1 v, E2=0.7v and E3=-0.1v) was used for detection. The HPLC chromatogram of PS1A showed three major peaks with retention times of 8.5, 15.5 and 19.25 min., respectively, and these three peaks have been identified in the subfractions of PS1A.