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.

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.

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.

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.

Nanomedicines and nanotoxicology: some physiological principles.

Nanosized materials have been investigated as potential medicines for several decades. Consequently, a great deal of work has been conducted on how to exploit constructs of this size range in a beneficial way. Similarly, a number of the consequences from the use of these materials have already been considered. Nanosized materials do behave differently to low-molecular-weight drugs, the biological properties of nanomaterials being mainly dependent on relevant physiology and anatomy, which are reviewed in this article. Biodistribution, movement of materials through tissues, phagocytosis, opsonization and endocytosis of nanosized materials are all likely to have an impact on potential toxicity. In turn these processes are most likely to depend on the nanoparticle surface. Evidence from the literature is considered which suggests that our understanding of these areas is incomplete, and that biodistribution to specific sites can occur for nanoparticles with particular characteristics. However, our current knowledge does indicate which areas are of concern and deserve further investigation to understand how individual nanoparticles behave and what toxicity may be expected from them.

The assessment of hookworm calreticulin as a potential vaccine for necatoriasis.

A vaccine against the human hookworm Necator americanus is urgently required to reduce hookworm-induced morbidity in endemic areas. In the present study, recombinant hookworm calreticulin, a nominated vaccine candidate, has been tested in mice. Mice given calreticulin had 43-49% fewer worms in their lungs, compared to non-vaccinated controls, following challenge infection with infective hookworm larvae. These levels of protection were achieved in the absence of adjuvant following intraperitoneal administration of three doses of 15 microg antigen. Antigen was also encapsulated in PLG microparticles. Encapsulated calreticulin elicited higher levels of anti-calreticulin IgG1 than free antigen but failed to induce protective immunity. The protection induced by free calreticulin was associated with low levels of serum IgE and moderate lung eosinophilia whilst administration of calreticulin-loaded microparticles was associated with high levels of serum IgE and higher lung eosinophil activity, suggesting that the classical Th2 phenotype may not always be associated with protective immunity, albeit in experimental necatoriasis.

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.

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.

Copolymers of amine methacrylate with poly(ethylene glycol) as vectors for gene therapy.

A series of structurally related copolymers of tertiary amine methacrylate with poly(ethylene glycol) (PEG) were investigated for their potential to serve as vectors for gene therapy. The effects of copolymer structure on the complexation and transfection ability were assessed. The ability of the PEG-based copolymers and DMAEMA homopolymer to bind and condense DNA was confirmed by gel electrophoresis, ethidium bromide displacement and transmission electron microscopy. The presence of PEG in the copolymers had a beneficial effect on their ability to bind to DNA. Colloidally stable complexes were obtained for all the PEG-copolymer systems as shown by uniformly discrete spherical images from transmission electron microscopy and approximate diameters of 80-100 nm by dynamic light scattering studies. DMAEMA homopolymer, however, produced agglomerated particles, confirming the important role played by the PEG chains in producing compact stable DNA complexes. Assessment of the effect of ionic strength of the buffer on the complexation and dissociation of the complexes indicated the importance of both electrostatic and non-electrostatic interactions in the polymer-DNA complexation. In vitro transfection experiments showed that DMAEMA homopolymer gave the highest level of transfection comparable to a control poly-L-lysine (PLL) system. The PEG-based copolymers gave reduced levels of transfection, most likely due to the steric stabilization effect of a PEG corona.

Determination of protection from serum nuclease activity by DNA-polyelectrolyte complexes using an electrophoretic method.

Polyelectrolyte complexes between cationic polymers and DNA have emerged as potential nonviral vectors for DNA delivery. For successful in vivo delivery, methods for analyzing their ability to prevent digestion of the DNA payload by serum nucleases are essential. We report here a simple assay to determine degradation of DNA in these complexes using standard electrophoretic techniques. The assay is based on a high pH buffer which can dissociate the complexes under standard electrophoretic conditions. This assay can be used qualitatively to determine the time taken for degradation to occur. Alternatively, with a standard gel analysis program it can be used quantitatively to investigate rates of DNA degradation from complexes in the presence of serum nucleases. We have shown that it can distinguish between different formulations with the same polymer, and also to distinguish between the time taken to degradation and the rates of degradation of DNA in complexes formed with two structurally related, linear polyamidoamine polymers. The assay could also distinguish between the time to degradation using poly-l-lysine complexes, although these were less well dissociated by the electrophoresis buffer, and could not be analyzed quantitatively. This assay will be of value in investigating and developing polyelectrolyte formulations for parenteral administration.

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.

Polylactide-poly(ethylene glycol) micellar-like particles as potential drug carriers: production, colloidal properties and biological performance.

The micellar-like particle systems produced from poly-D,L-lactide-poly(ethylene glycol) (PLA-PEG) copolymers have been assessed using a range of physicochemical characterisation methods, followed by in vivo studies of their biodistribution after intravenous administration to the rat. The size of the PEG chain was kept constant at 5 or 2 kDa, while the PLA size increased within a series from 2 to 25 kDa. The results obtained reveal, that in an aqueous medium the copolymers assembled into micellar-like structures, with the PLA segments forming the core and the PEG segments the surrounding corona. The size of the PLA segments dominated the process of assembly of the molecules and the characteristics of the resultant micellar-like particles. The PLA-PEG micellar particles were found to be less dynamic than those obtained from conventional surfactants. Particles formed from the lower molecular weight PLA polymers allowed a level of chain mobility while the cores of the micellar particles formed from higher molecular weight PLA appeared to be solid-like in nature. The size of the micellar particles was dependent on the copolymer molecular weight and the z-average diameter increased from 25 to 76 nm as the molecular weight of the PLA moiety increased. This provides an ability to control the particle size by adjusting the molecular weight of the PLA moiety. Following intravenous administration to the rat model, micellar-like particles smaller than approximately 70 nm accumulated in the liver, despite the fact that the PEG corona provided an effective steric stabilization effect. Micellar-like particles with a diameter of more than approximately 70 nm exhibited prolonged systemic circulation and reduced liver uptake, although the steric stabilisation of these particles was shown to be less effective. These findings agree with recent observations from other research groups; that indicate a possibility that very small particulates can pass through the sinusoidal fenestrations in the liver and gain access to the parenchymal cells of the liver.

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.

Defining the drug incorporation properties of PLA-PEG nanoparticles.

The drug incorporation and physicochemical properties of PLA-PEG micellar like nanoparticles were examined in this study using a model water soluble drug, procaine hydrochloride. Procaine hydrochloride was incorporated into nanoparticles made from a series of PLA-PEG copolymers with a fixed PEG block (5 kDa) and a varying PLA segment (3-110 kDa). The diameter of the PLA-nanoparticles increased from 27.7 to 174.6 nm, with an increase in the PLA molecular weight. However, drug incorporation efficiency remained similar throughout the series. Incorporation of drug into the smaller PLA-PEG nanoparticles made from 3:5, 15:5 and 30:5 copolymers did not influence the particle size, while an increase was observed for the larger systems comprising 75:5 and 110:5 copolymers. An increase in drug content for PLA-PEG 30:5 nanoparticles was achieved by increasing the theoretical loading (quantity of initially present drug). The size of these nanoparticles remained unchanged with the increasing drug content, supporting the proposed micellar type structure of the PLA-PEG 30:5 nanoparticles. The morphology of these systems remained unchanged both at low and high theoretical drug loadings. Formulation variables, such as an increase in the aqueous phase pH, replacement with the base form of the drug and inclusion of lauric acid in the formulation did not improve the incorporation efficiency of drug into PLA-PEG 30:5 nanoparticles. While poly(aspartic acid) as a complexation agent did not improve the drug incorporation efficiency of procaine hydrochloride, it did so for another water soluble drug diminazene aceturate. This may be attributed to a stronger interaction of diminazene aceturate with poly(aspartic acid) relative to procaine hydrochloride, as confirmed by thermodynamic analysis of isothermal titration calorimetric data. The drug incorporation and physicochemical characterisation data obtained in this study may be relevant in optimising the drug incorporation and delivery properties of these potential drug targeting carriers.

Preparation and in vitro characterization of HSA-mPEG nanoparticles.

Surface modified human serum albumin (HSA) nanoparticles with a size of approximately 150 nm in diameter were prepared from a PEG-HSA conjugate, methoxy-polyethylene glycol modified human serum albumin (HSA-mPEG) using a coacervation method and crosslinked with glutaraldehyde. The zeta-potential of the surface modified nanoparticles was significantly lower than that of unmodified HSA nanoparticles. The existence of a hydrated steric barrier surrounding the nanoparticles was confirmed by electrolyte and pH induced flocculation tests. The surface modified nanoparticles showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.

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.

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.

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.