The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes.

We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections.

The anthelmintic efficacy of papaya latex in a rodent-nematode model is not dependent on fasting before treatment.

In earlier studies of the anthelmintic activity of plant cysteine proteinases (CPs), a period of food deprivation was routinely employed before administration of CPs, but there has been no systematic evaluation as to whether this does actually benefit the anthelmintic efficacy. Therefore, we assessed the effect of fasting on the efficacy of CPs from papaya latex (PL) against Heligmosomoides bakeri in C3H mice. We used a refined, supernatant extract of papaya latex (PLS) with known active enzyme content. The animals were divided into three groups (fasted prior to treatment with PLS, not fasted but treated with PLS and fasted but given only water). The study demonstrated clearly that although food deprivation had been routinely employed in much of the earlier work on CPs in mice infected with nematodes, fasting has no beneficial effect on the efficacy of PLS against H. bakeri infections. Administration of CPs to fed animals will also reduce the stress associated with fasting.

Developing novel anthelmintics: the stability of cysteine proteinase activity in a supernatant extract of papaya latex.

Plant derived cysteine proteinases (CPs) have long been known to possess anthelmintic properties but have attracted renewed attention recently because of the acute need to discover novel methods for controlling helminth infections as a result of increasing drug resistance. However, surprisingly little is known about the stability of these proteins under typical storage and in vivo exposure conditions. We found that CPs in a supernatant preparation from papaya latex (PLS) were stable during the initial refinement process and when stored under low temperatures, but lost activity during dialysis and within 7 days of storage when kept at ambient temperature (18-20 °C). The enzyme activity in PLS was not affected by repeated freeze-thaw cycles and was also stable under typical in vitro assay conditions at 37 °C used for quantifying effects on helminths. Active enzyme activity was still detectable in the colon 3-4 h after oral administration in rodent models.

Evaluation of poly (glycerol-adipate) nanoparticle uptake in an in vitro 3-D brain tumor co-culture model.

Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.

Uptake and metabolism of novel biodegradable poly (glycerol-adipate) nanoparticles in DAOY monolayer.

A useful route for the development of antitumour therapies is by creating improved methods for delivering therapeutic agents to tumour cells or subcellular compartments and increasing retention of drugs within target cells. In this study, we have characterized nanoparticle (NP) uptake and metabolism by DAOY cells, a human medulloblastoma cell line. NPs were formed from a novel polymer, poly (glycerol-adipate) (PGA), containing Rhodamine B Isothiocyanate (RBITC) as a fluorescent marker. It was observed that the cellular uptake of NPs depends on the incubation time and the concentration of NPs in the culture medium. The studies of retention and metabolism of NPs within cells indicated that 1) faster degradation of NPs within cells compared with that in cell culture medium in vitro; 2) a small fraction of NPs were recycled back to the outside of cell, whereas most NPs entered endosomes and lysosomes; and 3) recycled NPs were re-taken up in the following 2 h incubation time. These studies thus suggested that PGA NPs could be used for localising therapeutic agents into cells, and could provide prolonged drug effects because of their long sustained release in physiological conditions and their rapid release when taken up into cells.

An improved synthesis of a methotrexate-albumin-791T/36 monoclonal antibody conjugate cytotoxic to human osteogenic sarcoma cell lines.

Improvements have been made in the synthesis of a drug-carrier-antibody conjugate using methotrexate as the drug, human serum albumin as the carrier, and a monoclonal antibody against a human osteogenic sarcoma cell line (791T/36). The improvements have resulted in a higher and more reproducible substitution of serum albumin by methotrexate, and improvements in the coupling of methotrexate covalently linked to human serum albumin to antibody resulting in a greater ease and efficiency of conjugation. The improvements have led to a conjugate of increased cytotoxicity while retaining the previously reported specificity. A conjugate is reported which shows cytotoxicity of 1.1 ng/ml (2.4 nM) with respect to methotrexate and 6 X 10(-11) M with respect to antibody in a clonogenic assay on 791T cells. This cytotoxicity is greater than that obtained using free methotrexate (2.8 ng/ml; 6.1 nM) and implies that drug cytotoxicity can be considered as the sum of drug uptake and the number of drug molecules required to kill a cell. This further suggests that antibodies could provide a potent delivery system for drugs which are poorly taken up by cells.

Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action.

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

In vitro cytotoxicity of poly(amidoamine)s: relevance to DNA delivery.

We have examined the cytotoxicity of a number of poly(amidoamine) polymers which have been proposed for use as DNA delivery systems and compared them to the charged polyamino acid polylysine. Most of the poly(amidoamine)s tested were shown to be remarkably non-toxic to both HepG2 and HL60 cell lines. However, one of the structures (NG30, co-monomers methylene bisacrylamide, dimethylethylene diamine) did show cytotoxicity similar to that of polylysine. A second PAA structure (NG37, NG38, NG39, co-monomers bisacryloyl piperazine, 2-methyl piperazine) showed mild cytotoxicity towards both cell lines, related to the degree of polymerisation. The results support the idea that the cytotoxicity of polycations has a strong structural basis rather than being an effect due only to charge. As a consequence of their general reduced level of cytotoxicity, poly(amidoamine)s appear to have possible advantages for complexation with DNA over some other cationic polymers as a key component of DNA delivery systems.

Polymer chemical structure is a key determinant of physicochemical and colloidal properties of polymer-DNA complexes for gene delivery.

Polyplexes are now emerging as potentially useful vectors for gene therapy. To improve our understanding of how the chemical structure of the polymer affects the properties of these systems, a series of structurally related polymers, the linear poly(amidoamine)s (PAAs), have been examined for their abilities to form complexes with DNA. Structure-dependent differences in DNA binding are shown by gel electrophoretic retardation of DNA and thermal transition analyses. Two PAAs, NG28 and NG30, stand out as having high affinity DNA binding characteristics, similar to the model homopolypeptide, poly-L-lysine. In addition, differences in complex formation, particle size and surface charge are displayed for the different polymer-DNA systems. Electron microscopy studies showed that the polymers condensed DNA into similar unit structures but only complexes with NG30 did not undergo agglomeration. This was attributed to an excess of complexed polymer forming a shell of uncomplexed polymer chain segments around a condensed DNA-polymer core. The transfection activities of these polymer complexes differ greatly, and some of these differences can be explained in a multifactorial way by the physicochemical and colloidal properties. It is concluded that polymer chemical structure dictates the apparent affinity of DNA binding, and also several of the important colloidal characteristics of the resulting complexes.

The effect of surface coverage and conformation of poly(ethylene oxide) (PEO) chains of poloxamer 407 on the biological fate of model colloidal drug carriers.

Poloxamer 407 was adsorbed onto the surface of model colloidal drug carriers, polystyrene nanoparticles of 40, 70 and 137 nm in diameter, and the effect of the degree of surface coverage and the conformation of the poly(ethylene oxide) (PEO) chains on biological fate was studied. The relationship between the physicochemical and the biological properties of the nanoparticle systems was also investigated. The adsorbed layer of poloxamer 407 was characterised in terms of percentage surface coverage, thickness of the adsorbed layer and average surface area per PEO chain. Computer modelling of the adsorbed layer was performed (applying the self-consistent field technique), to obtain the structural information of the PEO chains in the layer. The in vitro interaction of the nanoparticles with different degrees of poloxamer 407 surface coverage with serum components and the in vivo biodistribution in the rat model were assessed. The results demonstrated that an increase in the surface coverage with poloxamer 407 resulted in an increased volume fraction of the PEO in the adsorbed layer, further extension of the PEO chains from the surface and closer packing of the chains at the surface. With regard to the interaction with the serum components, an increased surface coverage resulted in a reduction of the amount of serum proteins adsorbed, and, importantly, affected the type of proteins adsorbed. High molecular weight proteins were not adsorbed onto the nanoparticles with a surface coverage above approx. 25%. Following the intravenous administration to rats, even the nanoparticles with the lowest degree of surface coverage (approx. 5%) showed improved circulation profiles relative to the uncoated nanoparticles. The effect was more pronounced for the 40 nm nanoparticles. A further increase in the surface coverage to approx. 25% resulted in a significant increase in circulation time, as compared to uncoated and 5% coated systems, for all sizes of nanoparticles. Importantly, it was found that a long in vivo blood circulation time could be achieved for nanoparticles with a relatively low degree of surface coverage with PEO chains.

Multiscale modeling of drug-polymer nanoparticle assembly identifies parameters influencing drug encapsulation efficiency.

Using a multiscale (dual resolution) approach combining an atomistic (GROMOS96) and coarse-grain (MARTINI) force field, we have been able to simulate the process of drug-polymer nanoparticle assembly by nanoprecipitation from mixed solvents. Here, we present the development and application of this method to the interaction of three poly(glycerol adipate) polymer variants with the anticancer drug dexamethasone phosphate. Differences in encapsulation efficiency and drug loading between the polymers are in agreement with the experimental trend. Reference atomistic simulations at key points along the predicted aggregation pathway support the accuracy of the much more computationally efficient multiscale methodology.

Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates.

The endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen has been investigated using several different fluorescent conjugates. These conjugates have been employed for both fluorescence microscopy to show the qualitative changes in distribution of antibody conjugates during endocytosis, and also flow cytofluorimetry to show the quantitative changes in fluorescence intensity associated with this redistribution. Using an antibody directly labelled with fluorescein it was difficult to demonstrate endocytosis due to the inability to distinguish clearly between internal and external fluorescence. However, a fluorescein-HSA-antibody conjugate which was heavily quenched at the cell surface was endocytosed and degraded during incubation at 37 degrees C for 4 h and was then visualised in a perinuclear distribution by the addition of agents to modify intracellular pH. This experiment demonstrated that such conjugates became localised within an acidic internal compartment. A tetramethylrhodamine-HSA-conjugate also demonstrated a similar perinuclear distribution but without the addition of endosomal pH modifiers. When used in conjunction with a fluorescein rabbit anti-HSA second label this conjugate also showed that not all conjugate was endocytosed during a 4-h period; some conjugate remained bound to the cell surface. These experiments suggested that endocytosis in this system differs from receptor-mediated endocytosis via coated pits which is reported to be more rapid and complete.

Targeted drug conjugates: principles and progress.

Reports of targeting drugs using antibodies have appeared in the literature since 1958, but exciting clinical results in this field have only been reported in the last few years. Progress in this field has occurred largely through an understanding how drug-immunoconjugates work. The objective of this review is to draw together the fundamental principles on which this field of work is based, to examine the evidence supporting those principles, and the effectiveness and selectivity of targeted drug conjugates. The activity of many drug-immunoconjugates can now largely be accounted for by the underlying principles. Excellent development work, both with conventional anti-cancer agents and very potent drugs have led to a number of interesting clinical trials. In the best Phase I and II trials, good evidence of effectiveness have been reported, which suggest that drug-immunoconjugates may now be heralding a new era for chemotherapy.

Mouse IgG2b monoclonal antibody fragmentation. Preparation and purification of Fab, Fc and Fab/c fragments.

Mouse IgG2b fragmentation has been poorly described in the literature because of the sensitivity of this subclass to proteases and the inability to produce F(ab')2 fragments. The fragments obtained include both Fc and Fab fragments and an intermediate of degradation, the Fab/c fragment, consisting of the Fc region and one Fab arm, which was first described by Parham (1983). Optimised pepsin digestion led to the formation of Fab/c in yields of up to 30% depending on the IgG2b antibodies susceptibility. DEAE-cellulose chromatography of the digested antibodies allowed, in all cases, separation of Fab fragments from Fc bearing fragments. Depending on the differences in pI between the fragments, Fab/c fragment purification was achieved either by CM-cellulose chromatography or by recycling HPLC gel filtration chromatography. Both procedures gave 97.5% purity Fab/c fragments. Fc fragments were purified by HPLC gel filtration chromatography. In cancer therapy the monovalent Fab/c fragments could be useful for drug targeting or for immunotherapy providing it retains a good affinity.

Synthesis of 2'-deoxyuridine and 5-fluoro-2'-deoxyuridine derivatives and evaluation in antibody targeting studies.

Derivatives of 2'-deoxyuridine and of the anticancer agent 5-fluoro-2'-deoxyuridine (FdUR) were linked indirectly via a human serum albumin carrier (HSA) to the murine antiosteosarcoma monoclonal antibody 791T/36. Starting from the 2'-deoxyuridines 1a and 1b, the new nucleosides containing 5'-succinamic acid 7 and 5'-maleamic acid 8 spacers were synthesized from the key intermediate 5'-aminonucleoside 4, and the ribofuronamidobenzoic acid 13 from ribofuranuronic acid 10. These nucleosides were linked via their spacer functionality to HSA. High molar substitution ratios (MSR: moles of drug/mole of HSA) of 25-40 for these derivative-HSA conjugates were achieved. All derivatives were less cytotoxic than the parent drug against both antigen positive osteogenic sarcoma 791T and antigen negative bladder carcinoma T24 cell lines; no IC50 was achieved with any derivative against 791T cells. The fluorodeoxyuridine-HSA conjugates were then further linked via a stable thioether bond to the mouse monoclonal antibody 791T/36. The optimum fluorinated 5'-succinamic acid immunoconjugate exhibited an IC50 of 1 microM against 791T and T24 cells, slightly better than that of fluorodeoxyuridine. The unconjugated derivative 7 was much less cytotoxic than immunoconjugate, with an IC50 of 62 microM on T24 cells, and failed to reach 50% inhibition of 791T cell growth at 290 microM concentration. Derivative 7-HSA conjugate was 10-fold less cytotoxic than the immunoconjugate against both cell lines. Immunoconjugates synthesized with the other 5-fluoro derivatives were unable to effect 50% inhibition of growth of cell lines. Nonfluorinated derivatives and their HSA conjugates and immunoconjugates exhibited no cytotoxicity.

Gene-delivery systems using cationic polymers.

Gene therapy will benefit a range of diseases from single-gene defects, to chronic diseases such as cancer, to vaccination. Initially, gene therapy used viral vectors, but the advantages of nonviral systems are now being fully appreciated. This review focuses on cationic polymers as a delivery system for DNA. The physicochemical characterization of DNA polycation complexes that condense and protect DNA from nuclease digestion are considered, together with further factors such as ligand targeting, endosomal escape, and nuclear localization. Where possible, the relative efficacy of different cationic polymer delivery systems is compared.

Preparation of surface-modified albumin nanospheres.

Surface-modified human serum albumin (HSA) nanospheres with a size of around 100 nm in diameter were prepared from poly(amidoamine)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PAA-PEG) and poly(thioetheramido acid)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PTAAC-PEG). The nanospheres were produced using a pH-coacervation method and cross-linked with glutaraldehyde. The cross-linking efficiency was affected by the type of albumin conjugate used. The zeta potential of the surface-modified nanospheres was significantly lower than that of unmodified particles. The existence of a hydrated steric barrier surrounding the nanospheres was confirmed by electrolyte- and pH-induced flocculation tests. The surface-modified nanospheres showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.

Long circulating biodegradable poly(phosphazene) nanoparticles surface modified with poly(phosphazene)-poly(ethylene oxide) copolymer.

The biodistribution of biodegradable poly(organo phosphazene) nanoparticles surface modified by adsorption of a novel poly(organo phosphazene)-poly(ethylene oxide) copolymer with a 5000 M(W) PEO chain (PF-PEO[5000]), following intravenous administration in rats and rabbits, is described. The data are compared to the biodistribution of poly(organo phosphazene) and poly(lactide-co-glycolide) nanoparticles coated with a tetrafunctional copolymer of poly(ethylene oxide)-poly(propylene oxide) ethylenediamine, commercially available as Poloxamine 908. This copolymer has a PEO chain of the same size as the poly(organo phosphazene)-PEO derivative used. The results in the rat model reveal that poly(organo phosphazene) nanoparticles with a Poloxamine 908 coating were mainly captured by the liver, although a retardation in clearance from the systemic circulation was seen. In contrast, the poly(organo phosphazene) nanoparticles coated with PF-PEO(5000) showed a prolonged blood circulating profile, with only a small amount of the nanoparticles sequestered by the liver. This indicates the importance of the nature of both the anchoring group and the particle surface on the biological performances of the system. Study of the biodistribution of the PF-PEO(5000)-coated poly(organo phosphazene) nanoparticles in the rabbit model also indicated a prolonged systemic circulation lifetime and reduced liver uptake, whereby a significant amount of the administered nanoparticles was targeted to the bone marrow.

PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug.

The nanoprecipitation technique for preparation of nanoparticles suffers the drawback of poor incorporation of water soluble drugs. The aim of this study was therefore to assess various formulation parameters to enhance the incorporation of a water soluble drug (procaine hydrochloride) into poly(dl-lactide-co-glycolide) (PLGA) nanoparticles prepared by this technique. Approaches investigated for drug incorporation efficiency enhancement included the influence of aqueous phase pH, replacement of procaine hydrochloride with procaine dihydrate and the inclusion of excipients: poly(dl-lactide) (PLA) oligomers, poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA) or fatty acids into the formulation. The nanoparticles produced were submicron size (<210 nm) and of low polydispersity. It was found that an aqueous phase pH of 9.3, replacement of procaine hydrochloride with procaine dihydrate and the incorporation of PMMA-MA, lauric and caprylic acid into the formulation could enhance drug incorporation efficiency without the size, morphology and nanoparticle recovery being adversely influenced. For instance changing the aqueous phase pH from 5.8 to 9.3 increased nanoparticle recovery from 65.1 to 93.4%, drug content from 0.3 to 1.3% w/w and drug entrapment from 11.0 to 58.2%. However, the presence of high ratios of lauric acid and procaine dihydrate in the formulation adversely affected the morphology and size of the nanoparticles. Also, PLA oligomers were not considered a feasible approach since it decreased drug entrapment from 11.0 to 8.4% and nanoparticle recovery from 65.1 to 19.6%. Drug release from nanoparticles appears to consist of two components with an initial rapid release followed by a slower exponential stage. This study has demonstrated that formulation variables can be exploited in order to enhance the incorporation of a water soluble drug into PLGA nanoparticles by the nanoprecipitation technique.

Preparation and characterisation of rose Bengal-loaded surface-modified albumin nanoparticles.

Surface-modified albumin nanoparticles were prepared from two poly(ethylene glycol)-human serum albumin conjugates: poly(thioetheramido acid)-poly(ethylene glycol) copolymer-grafted HSA (HSA-PTAAC-PEG) and methoxy poly(ethylene glycol)-grafted HSA (HSA-mPEG). Rose bengal (RB) was used as a model drug for encapsulation into the nanoparticles either during the particle production or by adsorption post particle preparation. The drug incorporation and release was affected by the different production methods and the different polymer compositions. When RB was loaded in HSA and HSA/HSA-PTAAC-PEG nanoparticles, up to 5% (w/w) drug content was achieved. The drug loading in HSA-mPEG nanoparticles was much lower and the results from the microcalorimetry study indicated that the low loading efficiency was due to less drug-protein binding sites available in the HSA-mPEG molecule as compared to the HSA molecule. The release of RB from the albumin nanoparticles was very slow in PBS and dramatically accelerated in the presence of trypsin. Compared with unmodified nanoparticles, the slower release of RB from the surface-modified HSA nanoparticles in the presence of the enzyme suggested that the existence of a steric hydrophilic barrier on the surface of the nanoparticles made digestion of the nanoparticles more difficult.