Coherent anti-Stokes Raman Scattering (CARS) Microscopy Visualizes Pharmaceutical Tablets During Dissolution

Traditional pharmaceutical dissolution tests determine the amount of drug dissolved over time by measuring drug content in the dissolution medium. This method provides little direct information about what is happening on the surface of the dissolving tablet. As the tablet surface composition and structure can change during dissolution, it is essential to monitor it during dissolution testing. In this work coherent anti-Stokes Raman scattering microscopy is used to image the surface of tablets during dissolution while UV absorption spectroscopy is simultaneously providing inline analysis of dissolved drug concentration for tablets containing a 50% mixture of theophylline anhydrate and ethyl cellulose. The measurements showed that in situ CARS microscopy is capable of imaging selectively theophylline in the presence of ethyl cellulose. Additionally, the theophylline anhydrate converted to theophylline monohydrate during dissolution, with needle-shaped crystals growing on the tablet surface during dissolution. The conversion of theophylline anhydrate to monohydrate, combined with reduced exposure of the drug to the flowing dissolution medium resulted in decreased dissolution rates. Our results show that in situ CARS microscopy combined with inline UV absorption spectroscopy is capable of monitoring pharmaceutical tablet dissolution and correlating surface changes with changes in dissolution rate.     

Adsorption of meloxicam on porous calcium silicate: Characterization and tablet formulation

The purpose of the present study was characterization of microparticles obtained by adsorption of poorly water soluble drug, meloxicam, on a porous silicate carrier Florite RE (FLR) and development of a tablet formulation using these microparticles, with improved drug dissolution properties. The study also reveals the use of FLR as a pharmaceutical excipient. Meloxicam was adsorbed on the FLR in 2 proportions (1∶1 and 1∶3), by fast evaporation of solvent from drug solution containing dispersed FLR. Drug adsorbed FLR microparticles were evaluated for surface topography, thermal analysis, X-ray diffraction properties, infrared spectrum, residual solvent, micromeritic properties, drug content, solubility, and dissolution studies. Microparticles showed bulk density in the range of 0.10 to 0.12 g/cm³. Dissolution of drug from microparticles containing 1∶3, drug∶FLR ratio was faster than microparticles containing 1∶1, drug∶FLR ratio. These microparticles were used for formulating directly compressible tablets. Prepared tablets were compared with a commercial tablet. All the prepared tablets showed acceptable mechanical properties. Disintegration time of prepared tablets was in the range of 18 to 38 seconds, and drug dissolution was much faster in both acidic and basic medium from prepared tablets as compared with commercial tablet. The results suggest that FLR provides a large surface area for drug adsorption and also that a reduction in crystallinity of drug occurs. Increase in surface area and reduction in drug crystallinity result in improved drug dissolution from microparticles. Published: December 7, 2005     

Utilizing inherent fluorescence of therapeutics to analyze real-time uptake and multi-parametric effector kinetics

The precise detection of pharmaceutical drug uptake and knowledge of a drug's efficacy at the single-cell level is crucial for understanding a compound's performance. Many pharmaceutical drugs, like the model substances Doxorubicin, Mitoxantrone or Irinotecan, have a distinctive natural fluorescence that can be readily exploited for research purposes. Utilizing this respective natural fluorescence, we propose a method analyzing simultaneously in real-time the efficiency, effects and the associated kinetics of compound-uptake and efflux in mammalian cells by flow cytometry. We show that real-time flow cytometric quantification of compound-uptake is reliably measured and that analyzing their respective uptake kinetic provides additional valuable information which can be used for improving drug dosage and delivery. Exploiting the native fluorescence of natural compounds is clearly advantageous compared to the usage of non-related fluorescent uptake-reporter substances, possibly yielding in unphysiological data. Flow cytometric analysis allows live-dye based multi-parametric high-throughput screening of pharmaceutical compound activity, improving cytotoxicity testing by combining several assays into a single, high resolution readout. This approach can be a useful tool identifying potential inhibitors for multiple drug resistance (MDR), representing a major challenge to the targeted treatment of various diseases.     

Novel molecules for intra-oral delivery of antimicrobials to prevent and treat oral infectious diseases

New molecules were designed for efficient intra-oral delivery of antimicrobials to prevent and treat oral infection. The salivary statherin fragment, which has high affinity for the tooth enamel, was used as a carrier peptide. This was linked through the side chain of the N-terminal residue to the C-terminus of a defensin-like 12-residue peptide to generate two bifunctional hybrid molecules, one with an ester linkage and the other with an anhydride bond between the carrier and the antimicrobial components. They were examined for their affinity to a HAP (hydroxyapatite) surface. The extent of the antimicrobial release in human whole saliva was determined using 13C-NMR spectroscopy. The candidacidal activity of the molecules was determined as a function of the antimicrobial release from the carrier peptide in human saliva. The hybrid-adsorbed HAP surface was examined against Candida albicans and Aggregatibacter actinomycetemcomitans using the fluorescence technique. The bifunctional molecules were tested on human erythrocytes, GECs (gingival epithelial cells) and GFCs (gingival fibroblast cells) for cytotoxicity. They were found to possess high affinity for the HAP mineral. In human whole saliva, a sustained antimicrobial release over a period of more than 40-60 h, and candidacidal activity consistent with the extent of hybrid dissociation were observed. Moreover, the bifunctional peptide-bound HAP surface was found to exhibit antimicrobial activity when suspended in clarified human saliva. The hybrid peptides did not show any toxic influence on human erythrocytes, GECs and GFCs. These novel hybrids could be safely used to deliver therapeutic agents intra-orally for the treatment and prevention of oral infectious diseases.     

Antimicrobial peptides with cell-penetrating peptide properties and vice versa

Antimicrobial peptides (AMPs) are a group of peptides that are active against a diverse spectrum of microorganisms. Due to their mode of action, AMPs are a promising class of molecules that could overcome the problems of increasing resistance of bacteria to conventional antibiotics. Furthermore, AMPs are strongly membrane-active and some are able to translocate into cells without the necessity for permanent membrane permeabilization. This feature has brought them into focus for use as transport vectors in the context of drug delivery. Since the plasma membrane restricts transport of bioactive substances into cells, great research interest lies in the development of innovative ways to overcome this barrier and to increase bioavailability. In this context, peptide-based transport systems, such as cell-penetrating peptides (CPPs), have come into focus, and their efficiency has been demonstrated in many different applications. However, more recently, also some AMPs have been used as efficient vectors for intracellular translocation of various active molecules. This review summarizes recent efforts in this interesting field of drug delivery. Moreover, some examples of the application of CPPs as efficient antimicrobial substances will be discussed.     

Raman characterization and chemical imaging of biocolloidal self-assemblies, drug delivery systems, and pulmonary inhalation aerosols: A review

This review presents an introduction to Raman scattering and describes the various Raman spectroscopy, Raman microscopy, and chemical imaging techniques that have demonstrated utility in biocolloidal self-assemblies, pharmaceutical drug delivery systems, and pulmonary research applications. Recent Raman applications to pharmaceutical aerosols in the context of pulmonary inhalation aerosol delivery are discussed. The "molecular fingerprint" insight that Raman applications provide includes molecular structure, drug-carrier/excipient interactions, intramolecular and intermolecular bonding, surface structure, surface and interfacial interactions, and the functional groups involved therein. The molecular, surface, and interfacial properties that Raman characterization can provide are particularly important in respirable pharmaceutical powders, as these particles possess a higher surface-area-to-volume ratio; hence, understanding the nature of these solid surfaces can enable their manipulation and tailoring for functionality at the nanometer level for targeted pulmonary delivery and deposition. Moreover, Raman mapping of aerosols at the micro- and nanometer level of resolution is achievable with new, sophisticated, commercially available Raman microspectroscopy techniques. This noninvasive, highly versatile analytical and imaging technique exhibits vast potential for in vitro and in vivo molecular investigations of pulmonary aerosol delivery, lung deposition, and pulmonary cellular drug uptake and disposition in unfixed living pulmonary cells.     

Improvisation and Evaluation of Laterosporulin Coated Titanium Surfaces for dental Applications: An In Vitro Investigation

Despite recent improvement in implant survival rates, there remains a significant demand for enhancing the long-term clinical efficacy of titanium (Ti) implants, particularly for the prevention of peri-implantitis. Bioactive substances such as antimicrobial peptides are emerging as effective alternatives for contemporary antimicrobial agents used in dental health care. Current research work was focused to use laterosporulins that are non-haemolytic cationic antimicrobial peptides from Brevibacillus spp. for coating commercially available Ti discs. The coated Ti surfaces were evaluated in vitro for biofilm formation by two dental plaque isolates Streptococcus gordonii strain DIGK25 and S. mutans strain DIGK119 as representatives of commensal and pathogenic streptococci respectively. The biofilm inhibition was ascertained with replicated experiments on hydroxyapatite discs and confirmed by florescence microscopy. The laterosporulin coated Ti discs showed significantly reduced biofilm formation by oral streptococci and displayed promising potential to enhance the antibacterial surface properties. Such improvised Ti surfaces may curb the menace of oral streptococcal biofilm formation on dental implants and the associated implant failures.     

Liposome as a delivery system for the treatment of biofilm-mediated infections

Biofilm formation by pathogenic microorganisms has been a tremendous challenge for antimicrobial therapies due to various factors. The biofilm matrix sequesters bacterial cells from the exterior environment and therefore prevents antimicrobial agents from reaching the interior. In addition, biofilm surface extracellular polymeric substances can absorb antimicrobial agents and thus reduce their bioavailability. To conquer these protection mechanisms, liposomes have been developed into a drug delivery system for antimicrobial agents against biofilm-mediated infections. The unique characteristics of liposomes, including versatility for cargoes, target-specificity, nonimmunogenicity, low toxicity, and biofilm matrix-/cell membrane-fusogenicity, remarkably improve the effectiveness of antimicrobial agents and minimize recurrence of infections. This review summarizes current development of liposomal carriers for biofilm therapeutics, presents evidence in their practical applications and discusses their potential limitations.     

Multilayer associates based on oligonucleotides and gold nanoparticles

The development of nanodevices for the efficient transport of therapeutic molecules is one of the most urgent problems of modern molecular medicine. Noncovalent agents for the delivery of nucleic acids (NA) including those based on gold nanoparticles (GNPs) represent an attractive alternative to covalent systems, since it is easier in this case to provide the controlled release of NA. We have demonstrated the possibility to create potentially biocompatible associates of GNPs containing alternating layers of oligonucleotides and other polymers as a promising platform for the delivery of oligonucleotides into living cells. The multilayer (five layers) coated GNPs can be assembled by the sequential treatment of gold nanoparticles with nonthiolated oligonucleotide (ON), thiolated carboxyl-polyethylene glycol (SH-PEG3000-COOH), and linear polyethyleneimine (PEI). We have developed an algorithm for the analysis of multilayer coated GNPs by gel electrophoresis, photon correlation spectroscopy, and transmission electron microscopy. The assembly of associates bearing two oligonucleotide layers and having a net positive surface charge has been described. Multilayer coated GNPs were shown not to degrade in the presence of a high concentration of the major blood protein, serum albumin.     

Biodegradable chitosan matrix for the controlled release of steroids.

Chitosan, a polysaccharide, having structural characteristics similar to glycosaminoglycans, seems to be nontoxic and bioabsorbable. This study highlights the use of chitosan matrix for controlled drug delivery systems. The steroid drugs, namely testosterone, progesterone and beta-oestradiol were mixed with chitosan and the films were prepared by evaporation technique. The in vitro release profile of these steroids from the film matrix was monitored, as a function of time, in phosphate buffered saline (PBS, pH 7.4) at 37 degree C using a U-V-spectrophotometer. The degradation, of these chitosan and drug loaded chitosan films, was also investigated by weight loss and tensile strength studies. The steroid release from chitosan films was compared with the release of these drugs from their microbeads. It appears, the films and the microbeads stayed intact during the dissolution study of 90 days and the possibility of using these systems in contraceptive applications and novel drug delivery systems are discussed.     

Sodium Montmorillonite/Amine-Containing Drugs Complexes: New Insights on Intercalated Drugs Arrangement into Layered Carrier Material

Layered drug delivery carriers are current targets of nanotechnology studies since they are able to accommodate pharmacologically active substances and are effective at modulating drug release. Sodium montmorillonite (Na-MMT) is a clay that has suitable properties for developing new pharmaceutical materials due to its high degree of surface area and high capacity for cation exchange. Therefore Na-MMT is a versatile material for the preparation of new drug delivery systems, especially for slow release of protonable drugs. Herein, we describe the intercalation of several amine-containing drugs with Na-MMT so we can derive a better understanding of how these drugs molecules interact with and distribute throughout the Na-MMT interlayer space. Therefore, for this purpose nine sodium montmorillonite/amine-containing drugs complexes (Na-MMT/drug) were prepared and characterized. In addition, the physicochemical properties of the drugs molecules in combination with different experimental conditions were assessed to determine how these factors influenced experimental outcomes (e.g. increase of the interlayer spacing versus drugs arrangement and orientation). We also performed a molecular modeling study of these amine-containing drugs associated with different Na-MMT/drug complex models to analyze the orientation and arrangement of the drugs molecules in the complexes studied. Six amine-containing drugs (rivastigmine, doxazosin, 5-fluorouracil, chlorhexidine, dapsone, nystatin) were found to successfully intercalate Na-MMT. These findings provide important insights on the interlayer aspect of the molecular systems formed and may contribute to produce more efficient drug delivery nanosystems.     

Nanoparticles,molecular biosensors,and multispectral confocal microscopy

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.     

Co-solvent Evaporation Method for Enhancement of Solubility and Dissolution Rate of Poorly Aqueous Soluble Drug Simvastatin: In vitro–In vivo Evaluation

A number of synthesized chemical molecules suffer from low aqueous solubility problems. Enhancement of aqueous solubility, dissolution rate, and bioavailability of drug is a very challenging task in drug development. In the present study, solubility and dissolution of poorly aqueous soluble drug simvastatin (SIM) was enhanced using hydrophilic, low viscosity grade polymer hydroxypropyl methylcellulose (HPMC K₃LV). The co-solvent evaporation method was developed for efficient encapsulation of hydrophobic drug in polymer micelles of HPMC K₃LV. Spray drying and rotaevaporation method were applied for solvent evaporation. Co-solvent-evaporated mixture in solid state was determined by differential scanning calorimetry (DSC), X-ray diffraction studies (XRD), scanning electron microscopy, and Fourier-transform infrared spectroscopy. In vitro–in vivo studies were performed on co-solvent-evaporated mixture and compared with SIM. In vivo study was conducted on healthy albino rats (Wister strain), and formulations were administered by oral route. Results of the study show the conversion of crystalline form of SIM into amorphous form. The dissolution rate was remarkably increased in co-solvent-evaporated mixtures compared to SIM. co-solvent-evaporated mixtures showed better reduction in total cholesterol and triglyceride levels than the SIM. The low-viscosity grade HPMC acts as a surfactant, which enhances the wetting of drug and thus improves the solubility of drug. The co-solvent evaporation method provides good encapsulation efficiency and produces amorphous form of SIM, which gave better solubility and dissolution than the crystalline SIM.     

Preparation of Chloramphenicol/Amino Acid Combinations Exhibiting Enhanced Dissolution Rates and Reduced Drug-Induced Oxidative Stress

Chloramphenicol is an old antibiotic agent that is re-emerging as a valuable alternative for the treatment of multidrug-resistant pathogens. However, it exhibits suboptimal biopharmaceutical properties and toxicity profiles. In this work, chloramphenicol was combined with essential amino acids (arginine, cysteine, glycine, and leucine) with the aim of improving its dissolution rate and reduce its toxicity towards leukocytes. The chloramphenicol/amino acid solid samples were prepared by freeze-drying method and characterized in the solid state by using Fourier transform infrared spectroscopy, powder X-ray diffraction, differential scanning calorimetry, scanning electron microscopy, and solid-state nuclear magnetic resonance. The dissolution properties, antimicrobial activity, reactive oxygen species production, and stability of the different samples were studied. The dissolution rate of all combinations was significantly increased in comparison to that of the pure active pharmaceutical ingredient. Additionally, oxidative stress production in human leukocytes caused by chloramphenicol was decreased in the chloramphenicol/amino acid combinations, while the antimicrobial activity of the antibiotic was maintained. The CAP:Leu binary combination resulted in the most outstanding solid system makes it suitable candidate for the development of pharmaceutical formulations of this antimicrobial agent with an improved safety profile.     

Drug delivery and nanoparticles:applications and hazards

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Interestingly pharmaceutical sciences are using nanoparticles to reduce toxicity and side effects of drugs and up to recently did not realize that carrier systems themselves may impose risks to the patient. The kind of hazards that are introduced by using nanoparticles for drug delivery are beyond that posed by conventional hazards imposed by chemicals in classical delivery matrices. For nanoparticles the knowledge on particle toxicity as obtained in inhalation toxicity shows the way how to investigate the potential hazards of nanoparticles. The toxicology of particulate matter differs from toxicology of substances as the composing chemical(s) may or may not be soluble in biological matrices, thus influencing greatly the potential exposure of various internal organs. This may vary from a rather high local exposure in the lungs and a low or neglectable exposure for other organ systems after inhalation. However, absorbed species may also influence the potential toxicity of the inhaled particles. For nanoparticles the situation is different as their size opens the potential for crossing the various biological barriers within the body. From a positive viewpoint, especially the potential to cross the blood brain barrier may open new ways for drug delivery into the brain. In addition, the nanosize also allows for access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently under investigation for the preparation of nanoparticles for drug delivery, varying from biological substances like albumin, gelatine and phospholipids for liposomes, and more substances of a chemical nature like various polymers and solid metal containing nanoparticles. It is obvious that the potential interaction with tissues and cells, and the potential toxicity, greatly depends on the actual composition of the nanoparticle formulation. This paper provides an overview on some of the currently used systems for drug delivery. Besides the potential beneficial use also attention is drawn to the questions how we should proceed with the safety evaluation of the nanoparticle formulations for drug delivery. For such testing the lessons learned from particle toxicity as applied in inhalation toxicology may be of use. Although for pharmaceutical use the current requirements seem to be adequate to detect most of the adverse effects of nanoparticle formulations, it can not be expected that all aspects of nanoparticle toxicology will be detected. So, probably additional more specific testing would be needed.     

Romping the cellular landscape: linear scaffolds for molecular recognition

Multivalent molecules with a precise array of recognition elements that interact with specific cell types are important for characterizing the topology of molecules on a cell surface. Applications ranging from the control of cellular signaling to drug delivery and tissue imaging rely on these surface-mapping molecules. Linear polymers provide a molecular scaffold that is advantageous for these types of applications and their synthesis can be amenable to the introduction of different recognition elements. Recently, advances have been made in the development of synthetic approaches for preparing linear polymeric substrates with highly controlled lengths and recognition element spacing.     

Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Applications of ATR-FTIR spectroscopic imaging to biomedical samples

FTIR spectroscopic imaging in ATR (Attenuated Total Reflection) mode is a powerful tool for studying biomedical samples. This paper summarises recent advances in the applications of ATR-FTIR imaging to dissolution of pharmaceutical formulations and drug release. The use of two different ATR accessories to obtain chemical images of formulations in contact with water as a function of time is demonstrated. The innovative use of the diamond ATR accessory allowed in situ imaging of tablet compaction and dissolution. ATR-FTIR imaging was also applied to obtain images of the surface of skin and the spatial distribution of protein and lipid rich domains was obtained. Chemical images of cross-section of rabbit aorta were obtained using a diamond ATR accessory and the possibility of in situ imaging of arterial samples in contact with aqueous solution was demonstrated for the first time. This experiment opens an opportunity to image arterial samples in contact with solutions containing drug molecules. This approach may help in understanding the mechanisms of treatment of atherosclerosis.     

Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners

The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.     

Can antimicrobial peptides scavenge around a cell in less than a second?

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide-lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use ¹⁵N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water-lipid interface.