Maximiliano L. Cacicedo

Progress in bacterial cellulose matrices for biotechnological applications

Bacterial cellulose (BC) is an extracellular polymer produced by many microorganisms. The Komagataeibacter genus is the best producer using semi-synthetic media and agricultural wastes. The main advantages of BC are the nanoporous structure, high water content and free hydroxyl groups. Modification of BC can be made by two strategies: in-situ, during the BC production, and ex-situ after BC purification. In bioprocesses, multilayer BC nanocomposites can contain biocatalysts designed to be suitable for outside to inside cell activities. These nanocomposites biocatalysts can (i) increase productivity in bioreactors and bioprocessing, (ii) provide cell activities does not possess without DNA cloning and (iii) provide novel nano-carriers for cell inside activity and bioprocessing. In nanomedicine, BC matrices containing therapeutic molecules can be used for pathologies like skin burns, and implantable therapeutic devices. In nanoelectronics, semiconductors BC-based using salts and synthetic polymers brings novel films showing excellent optical and photochemical properties.

Synthesis and characterization of CaCO3–biopolymer hybrid nanoporous microparticles for controlled release of doxorubicin

Doxorubicin (Dox) is a hydrophilic drug extensively used for treatment of breast, lung, and ovarian cancer, among others; it is highly toxic and can cause serious side effects on nontargeted tissues. We developed and studied a hybrid nanoporous microparticle (hNP) carrier based on calcium carbonate and biopolymers derivatized with folic acid (FA) and containing Dox as a chemotherapeutic drug model. The hNPs were characterized by X-ray diffraction, and Raman and Fourier transform infrared (FTIR) spectroscopies. The X-ray diffraction patterns of calcium carbonate particles showed about 30-70% vaterite-calcite polymorphisms and up to 95% vaterite, depending on the absence or the presence of biopolymers as well as their type. Scanning electron microcopy images revealed that all types of hNPs were approximately spherical and porous with average diameter 1-5 μm, and smaller than CaCO3 microparticles. The presence of biopolymers in the matrices was confirmed after derivatization with a fluorescein isothiocyanate probe by means of confocal microscopy and FTIR synchrotron beamline analysis. In addition, the coupling of lambda carrageenan (λ-Car) to FA in the microparticles (FA-λ-Car-hNPs) increased the cancer-cell targeting and also extended the specific surface area by up to ninefold (26.6 m2 g(-1)), as determined by the Brunauer-Emmett-Teller isotherm. A nanostructured porous surface was found in all instances, and the FA-λ-Car-hNP pore size was about 30 nm, as calculated by means of the Barrett-Joyner-Halenda adsorption average. The test of FA-λ-Car-hNP anticancer activity on human osteosarcoma MG-63 cell line showed cell viabilities of 13% and 100% with and without Dox, respectively, as determined by crystal violet staining after 24 h of incubation.

Modified bacterial cellulose scaffolds for localized doxorubicin release in human colorectal HT-29 cells.

Bacterial cellulose (BC) films modified by the in situ method with the addition of alginate (Alg) during the microbial cultivation of Gluconacetobacter hansenii under static conditions increased the loading of doxorubicin by at least three times. Biophysical analysis of BC-Alg films by scanning electron microscopy, thermogravimetry, X-ray diffraction and FTIR showed a highly homogeneous interpenetrated network scaffold without changes in the BC crystalline structure but with an increased amorphous phase. The main molecular interactions determined by FTIR between both biopolymers clearly suggest high compatibility. These results indicate that alginate plays a key role in the biophysical properties of the hybrid BC matrix. BC-Alg scaffold analysis by nitrogen adsorption isotherms revealed by the Brunauer-Emmett-Teller (BET) method an increase in surface area of about 84% and in pore volume of more than 200%. The Barrett-Joyner-Halenda (BJH) model also showed an increase of about 25% in the pore size compared to the BC film. Loading BC-Alg scaffolds with different amounts of doxorubicin decreased the cell viability of HT-29 human colorectal adenocarcinoma cell line compared to the free Dox from around 95-53% after 24h and from 63% to 37% after 48 h. Dox kinetic release from the BC-Alg nanocomposite displayed hyperbolic curves related to the different amounts of drug payload and was stable for at least 14 days. The results of the BC-Alg nanocomposites show a promissory potential for anticancer therapies of solid tumors.

Nanopharmaceuticals as a solution to neglected diseases: Is it possible?

The study of neglected diseases has not received much attention, especially from public and private institutions over the last years, in terms of strong support for developing treatment for these diseases. Support in the form of substantial amounts of private and public investment is greatly needed in this area. Due to the lack of novel drugs for these diseases, nanobiotechnology has appeared as an important new breakthrough for the treatment of neglected diseases. Recently, very few reviews focusing on filiarasis, leishmaniasis, leprosy, malaria, onchocerciasis, schistosomiasis, trypanosomiasis, and tuberculosis, and dengue virus have been published. New developments in nanocarriers have made promising advances in the treatment of several kinds of diseases with less toxicity, high efficacy and improved bioavailability of drugs with extended release and fewer applications. This review deals with the current status of nanobiotechnology in the treatment of neglected diseases and highlights how it provides key tools for exploring new perspectives in the treatment of a wide range of diseases.

Construction and in vitro testing of a cellulose dura mater graft

Introduction: Cerebrospinal fluid (CSF) leaks are a common complication after cranial and spinal surgery and are associated with increased morbidity. Despite continuous research in this field, this problem is far from solved. In this paper, we describe the construction and testing of a bacterial cellulose (BC) membrane as a new dural patch. Materials and Methods: The synthesis of BC was performed using Gluconacetobacter hansenii (ATCC 23769) and films were sterilized by autoclaving. The membranes were seeded with human dural fibroblasts. Growth, shape, and cell viability were assessed after 4 weeks. Results: Normally shaped fibroblasts were seen on the BC grafts; confocal microscopy showed cells inside the structure of the mesh. Both viable and nonviable cells were present. Cellular attachment and viability were confirmed by replating of the membranes. Discussion: BC membranes are used in clinical practice to improve skin healing. In the presence of water, they form an elastic, nontoxic, and resistant biogel that can accommodate collagen and growth factors within their structure, thus BC is a good candidate for dural graft construction.

Hybrid bacterial cellulose–pectin films for delivery of bioactive molecules

Novel biopolymeric films based on bacterial cellulose (BC) modified with high methoxylated pectin (HMP) were developed for drug delivery. The ability of the films to incorporate an antibiotic, levofloxacin (Levo), was analyzed. Incorporation efficiencies were determined using films with different proportions of HMP (from 0.1% to 2.0%) with a maximum drug payload of 6.23 mg g−1. Characterization studies revealed the existence of a cooperative network between both polymers and deep structural changes in the BC matrix. Besides, the presence of HMP decreased water loss in the BC films from 93% to 75% after 90 min. Additionally, the capacity of the films to incorporate macromolecules was studied using Human Serum Albumin (HSA) as a model protein. The presence of HMP enhanced by more than 3.5 times the encapsulation efficiency (EE) of HSA, and no pH dependence was observed. Release kinetics of both molecules showed hyperbolic profiles with sustained release. In independent experiments, the presence of HMP generated a decrease of around 50% in the release rates of both macromolecules. Additionally, the incorporation of HSA into the BC–HMP matrix modulated the Levo release profile. The antimicrobial activity of Levo released from the BC–HMP–HSA films was confirmed using Staphylococcus aureus. In vitro studies revealed no apparent cytotoxicity of the released compounds in mammalian CHO cells.

Development and Tailoring of Hybrid Lipid Nanocarriers

BACKGROUND Lipid nanoparticles are considered one of the most promising systems for controlled release of therapeutic molecules highly hydrophobic and with low biodisponibility. Solid lipid nanoparticles and nanostructured lipids carriers are widely seen as the workhorses of drug delivery systems because of low toxicity, enhanced encapsulation capacity, controlled drug kinetic release, easy tailoring and targeting and practicable scale up. CONCLUSIONS A new generation of hybrid lipid nanoparticles has emerged by combining the lipidic properties with polymers, proteins and metallic structures. The main features of hybrid lipid nanoparticles including popular methods for synthesis and characterization, biological and toxicological properties, administration routes, drug encapsulation strategies, tailoring and targeting, and potential systems for use in biomedicine are described in the present review.

Bacterial cellulose hydrogel loaded with lipid nanoparticles for localized cancer treatment

The use of hybrid materials, where a matrix sustains nanoparticles controlling the release of the chemotherapeutic drug, could be beneficial for the treatment of primary tumors prior or after surgery. This localized chemotherapy would guarantee high drug concentrations at the tumor site while precluding systemic drug exposure minimizing undesirable side effects. We combined bacterial cellulose hydrogel (BC) and nanostructured lipid carriers (NLCs) including doxorubicin (Dox) as a drug model. NLCs loaded with cationic Dox (NLCs-H) or neutral Dox (NLCs-N) were fully characterized and their cell internalization and cytotoxic efficacy were evaluated in vitro against MDA-MB-231 cells. Thereafter, a fixed combination of NLCs-H and NLCs-N loaded into BC (BC-NLCs-NH) was assayed in vivo into an orthotopic breast cancer mouse model. NLCs-H showed low encapsulation efficiency (48%) and fast release of the drug while NLCs-N showed higher encapsulation (97%) and sustained drug release. Both NLCs internalized via endocytic pathway, while allowing a sustained release of the Dox, which in turn rendered IC50 values below of those of free Dox. Taking advantage of the differential drug release, a mixture of NLCs-N and NLCs-H was encapsulated into BC matrix (BC-NLCs-NH) and assayed in vivo, showing a significant reduction of tumor growth, metastasis incidence and local drug toxicities.

BaCarb™: anovel bioinorganic matrix for local drug delivery

Background Treatment of localized tumor by chemotherapy is mostly used as an adjuvant to surgery to protect against or delay the progression of disseminated metastatic diseases or for treatment when other local therapies, such as surgery are not feasible. Local drug delivery offers high potential as a therapeutic choice against premature oncological stages or with isolated cancers since it is more effective in reducing cancer recurrences and avoiding the undesirable side effects of drug spreading along the body [1]. Several applications of the bacterial cellulose in the biomedical field were proposed, like wound healing membranes based on the similarities to the human skin, artificial blood vessels, and as replacement of cornea, valve prosthesis, urethra, cartilage, etc. Also, the use of cellulose membranes has been postulated for drug delivery of hormones and proteins and it was recently fully reviewed [2,3]. Doxorubicin hydrochloride (dox) is commonly used in the treatment of many solid tumors, such as breast, lung, stomach or ovarian cancer and sarcoma [4]. In the present work, we develop a bacterial cellulose membrane for encapsulation of Doxorubicin and containing l-Carr and CaCO3 as a potential implant device for local treatment of solid tumors. The in vitro Dox release kinetics from the matrices was studied at different pH values and also in different matrices. Methods The system of bacterial cellulose containing hybrid microparticles (HMPs) was carried out by colloidal crystallization in the presence of CaCl2 ,g lycine (Gly), Na2CO3 and l-carrageenan (Carr) [5]. To evaluate the potential for incorporation of chemotherapy, the hybrid system was immersed in a doxorubicin solution (Dox). The in vitro kinetics was carried out in PBS buffer. Results end conclusions The developed systems have the advantages of being synthesized on bacterialcellulose, a biomaterial broadly used in many biomedicalengineering applications, where cellulose is used as a physical support and a biological scaffold for tissue formation. Addition of Carr to the matrix raises the Dox loading only to 16.6%. However, the presence of CaCO3 raises up to 83.3% and 87.3% the Dox loaded into BC and BC-Carr, respectively. The presence of HMPs attached to MC allowed the increase of the Dox loading and made strict control of the drug release produced by local changes of pH made by tumoral cells. The Dox administration can be modulated by the amount of Dox required in the treatment and also by the amount of CaCO3 and their crystallization conditions. The system has the simplicity of Dox encapsulation in the HMPs by a technical procedure that allows to establish the amount of Dox/matrix required for specific treatment. The high loading capability of the Dox-CaCO3-Carr-BC matrix is advantageous since it is reducing the drug waste. Finally, the biodegradable matrix could be used for the treatment of oncological pathologies, particularly for breast cancer by local delivery of doxorubicin and defining the amount of drug to be released.