The modification of PLA and PLGA using electron-beam radiation

The degradable polymers polylactide (PLA) and polylactide-co-glycolide (PLGA) have found widespread use in modern medical practice. However, their slow degradation rates and tendency to lose strength before mass have caused problems. The aim of this study was to ascertain whether treatment with e-beam radiation could address these problems. Samples of PLA and PLGA were manufactured and placed in layered stacks, 8.1 mm deep, before exposure to 50 kGy of e-beam radiation from a 1.5 MeV accelerator. Gel permeation chromatography testing showed that the molecular weight of both materials was depth-dependent following irradiation, with samples nearest to the treated surface showing a reduced molecular weight. Samples deeper than 5.4 mm were unaffected. Computer modeling of the transmission of a 1.5 MeV e-beam in these materials corresponded well with these findings. An accelerated mass-loss study of the treated materials found that the samples nearest the irradiated surface initiated mass loss earlier, and at later stages showed an increased percentage mass loss. It was concluded that e-beam radiation could modify the degradation of bioabsorbable polymers to potentially improve their performance in medical devices, specifically for improved orthopedic fixation.

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

In this present work we describe a poly(lactic-co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells. (C) 2010 Elsevier Ltd. All rights reserved.

Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immunonanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 mu g coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT 116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC50 of 21.8 ng mL(-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC50 of 0.37 ng mL(-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

Cationic Bovine Serum Albumin (CBA) conjugated Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles for extended delivery of methotrexate into brain tumor

Current treatment strategies for the treatment of brain tumor have been hindered primarily by the presence of highly lipophilic insurmountable blood-brain barrier (BBB). The purpose of current research was to investigate the efficiency of engineered biocompatible polymeric nanoparticles (NPs) as drug delivery vehicle to bypass the BBB and enhance biopharmaceutical attributes of anti-metabolite methotrexate (MTX) encapsulated NPs. The NPs were prepared by solvent diffusion method using cationic bovine serum albumin (CBA), and characterized for physicochemical parameters such as particle size, polydispersity index, and zeta-potential; while the surface modification was confirmed by FTIR, and NMR spectroscopy. Developed NPs exhibited zestful relocation of FITC tagged NPs across BBB in albino rats. Further, hemolytic studies confirmed them to be non-toxic and biocompatible as compared to free MTX. In vitro cytotoxicity assay of our engineered NPs on HNGC1 tumor cells proved superior uptake in tumor cells; and elicited potent cytotoxic effect as compared to plain NPs and free MTX solution. The outcomes of the study evidently indicate the prospective of CBA conjugated poly (D,L-lactide-co-glycolide) (PLGA) NPs loaded with MTX in brain cancer bomber with amplified capability to circumvent BBB.

Enhanced surface attachment of protein-type targeting ligands to poly(lactide-co-glycolide) nanoparticles using variable expression of polymeric acid functionality

The density of reactive carboxyl groups on the surface of poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) was modulated using a combination of high-molecular weight (MW) encapped and low MW non-encapped PLGA. Carboxyl groups were activated using carbodiimide chemistry and conjugated to bovine serum albumin and a model polyclonal antibody. Activation of carboxyl,groups in solution-phase PLGA prior to NP formation was compared with a postformation activation of peripheral carboxyl groups on intact NP. Activation before or after NP formation did not influence conjugation efficiency to NP prepared using 100% of the low-MW PLGA. The effect of steric stabilization using poly(vinyl alcohol) reduced conjugation of a polyclonal antibody from 62 mu g/(mg NP) to 32 mu g/(mg NP), but enhanced particulate stability. Increasing the amount of a high-MW PLGA also reduced Conjugation, with the activation post-formation still superior to the preformation approach. Drug release studies showed that high proportions of high-MW PLGA in the NP produced a longer sustained release profile of a model drug (celecoxib). It can be concluded that activating intact PLGA NP is superior to activating component parts prior to NP formation. Also, high MW PLGA could be used to prolong drug release, but at the expense of conjugation efficiency on to the NP surface. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 873-884, 2008

Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Electron-beam treatment of poly(lactic acid) to control degradation profiles

Bioresorbable polymers such as polylactide (PIA) and polylactide-co-glycolide (PLGA) have been used successfully as biomaterials in a wide range of medical applications. However, their slow degradation rates and propensity to lose strength before mass have caused problems. A central challenge for the development of these materials is the assurance of consistent and predictable in vivo degradation. Previous work has illustrated the potential to influence polymer degradation using electron beam (e-beam) radiation. The work addressed in this paper investigates further the utilisation of e-beam radiation in order to achieve a more surface specific effect. Variation of e-beam energy was studied as a means to control the effective penetrative depth in poly-L-lactide (PLEA). PLEA samples were exposed to e-beam radiation at individual energies of 0.5 MeV, 0.75 MeV and 1.5 MeV. The near-surface region of the PLEA samples was shown to be affected by e-beam irradiation with induced changes in molecular weight, morphology, flexural strength and degradation profile. Moreover, the depth to which the physical properties of the polymer were affected is dependent on the beam energy used. Computer modelling of the transmission of each e-beam energy level used corresponded well with these findings. (C) 2010 Elsevier Ltd. All rights reserved.

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery

Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality. (C) 2011 Elsevier Ltd. All rights reserved.

The potential of polymeric nanoparticles to increase the immunogenicity of the hapten clenbuterol

Micro-and nanoparticles prepared front the biodegradable and biocompatible polymers poly(lactide-co-glycolide) (PLGA) and polymetylmethacrylate (PMMA) have been successfully used as immunopotentiating antigen delivery systems. In our study, this approach was used to improve polyclonal antibody production to clenbuterol (CBL), a model hapten. PLGA and PMMA nanoparticles were loaded with either CBL alone or with a clenbuterol-transferrin conjugate (CBL-Tfn) and administered subcutaneously to mice. PLGA nano-particles were administered with or without the saponin adjuvant Quil A. The anti-CBL titres present in experimental sera were determined by an enzyme immunoassay (ELISA). CBL-Tfn-loaded PLGA nanoparticles co-administered with Quil A had obvious advantages immmunologically over the currently used method of raising antibodies to CBL (the positive control). The combined adjuvanticity of Quil A and PLGA nanoparticles resulted in a positive response in all four of the mice tested and in higher antibody titles than were seen in the positive control group. Furthermore, the sustained release of immunogen from the nanoparticles permitted a reduction in immunizing frequency over the 15-week study period.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

The potential of electron beam radiation for simultaneous surface modification and bioresorption control of PLLA

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the fields of tissue engineering and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic acid (PLLA), Llactide/DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic acid (PLGA)). This work investigates the further potential of ebeam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. PLLA samples were subjected to e-beam treatments in air, with varying beam energies and doses. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy. Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA (as predicted). However, e-beam effects on surface properties were not shown to be dependent on beam energy or dose. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment.

Gentamicin-loaded nanoparticles show improved antimicrobial effects towards Pseudomonas aeruginosa infection

Gentamicin is an aminoglycoside antibiotic commonly used for treating Pseudomonas infections, but its use is limited by a relatively short half-life. In this investigation, developed a controlled-release gentamicin formulation using poly(lactide-co-glycolide) (PLGA) nanoparticles. We demonstrate that entrapment of the hydrophilic drug into a hydrophobic PLGA polymer can be improved by increasing the pH of the formulation, reducing the hydrophilicity of the drug and thus enhancing entrapment, achieving levels of up to 22.4 µg/mg PLGA. Under standard incubation conditions, these particles exhibited controlled release of gentamicin for up to 16 days. These particles were tested against both planktonic and biofilm cultures of P. aeruginosa PA01 in vitro, as well as in a 96-hour peritoneal murine infection model. In this model, the particles elicited significantly improved antimicrobial effects as determined by lower plasma and peritoneal lavage colony-forming units and corresponding reductions of the surrogate inflammatory indicators interleukin-6 and myeloperoxidase compared to free drug administration by 96 hours. These data highlight that the controlled release of gentamicin may be applicable for treating Pseudomonas infections.

Microneedle/nanoencapsulation-mediated transdermal delivery:Mechanistic insights

A systematic study was undertaken to gain more insight into the mechanism of transdermal delivery of nanoencapsulated model dyes across microneedle (MN)-treated skin, a complex process not yet explored. Rhodamine B (Rh B) and fluorescein isothiocyanate (FITC) as model hydrophilic and hydrophobic small/medium-size molecules, respectively, were encapsulated in poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and delivered through full thickness porcine skin pretreated with MN array. Permeation through MN-treated skin was affected by physicochemical characteristics of NPs and the encapsulated dyes. Dye flux was enhanced by smaller particle size, hydrophilicity, and negative zeta potential of NPs. Regarding encapsulated dyes, solubility at physiological pH and potential interaction with skin proteins proved to outweigh molecular weight as determinants of skin permeation. Data were verified using confocal laser scanning microscopy imaging. Findings coupled with the literature data are supportive of a mechanism involving influx of NPs, particularly of smaller size, deep into MN-created channels, generating depot dye-rich reservoirs. Molecular diffusion of the released dye across viable skin layers proceeds at a rate determined by its molecular characteristics. Data obtained provide mechanistic information of importance to the development of formulation strategies for more effective intradermal and transdermal MN-mediated delivery of nanoencapsulated therapeutic agents.