Applicability of fluorescamine as a fluorogenic reagent for highly sensitive fluorimetric analysis of the tyrosine kinase inhibitor (avapritinib) in pharmaceuticals and biological samples.

Avapritinib (AVP) was the first precision drug to be approved by the US Food and Drug Administration (FDA) in 2020 for patients suffering from metastatic gastrointestinal stromal tumors (GISTs) and progressive systemic mastocytosis. The analysis of AVP in pharmaceutical tablets and human plasma was then carried out using a fast, efficient, sensitive, and simple fluorimetric method using a fluorescamine reagent. The procedure is based on the interaction between fluorescamine as a fluorogenic reagent and the primary aliphatic amine moiety in AVP using borate buffer solution at pH 8.8. The produced fluorescence was measured at 465 nm (Excitation at 395 nm). The calibration graph's linearity range was discovered to be 45.00-500.0 ng mL-1 . Utilizing the International Council for Harmonization (ICH) and US-FDA recommendations, the research technique was validated and bioanalytically validated. The proposed approach was effectively employed for determining the stated pharmaceuticals in plasma with a high percentage of recovery ranging from 96.87 to 98.09 and pharmaceutical formulations with a percentage of recovery equal to 102.11% ± 1.05%. In addition, the study was extended to a pharmacokinetic study of AVP with 20 human volunteers as a step for AVP management in therapeutic cancer centers.

A feasible fluorimetric approach anchored in diaryl pyrrolone derivative for the facile analysis of milnacipran in tablets; evaluation of the method greenness.

In the present work a new, feasible, and green approach was employed for the analysis of milnacipran. A drug is used in the management of depression in addition to fibromyalgia. It inhibits of the reuptake of two essential neurotransmitters serotonin and nor-adrenaline. In slightly alkaline buffer (pH 8.5) the primary amino group of milnacipran reacted with fluorescamine to give a substituted pyrrolone derivative which exhibited high fluorescence activity. The fluorescence of produced derivative was measured at (λex 385 nm, λem 477 nm), and the experimental factors were cautiously optimized. The measured intensity of fluorescence was plotted versus the respective concentration of milnacipran to setup the calibration plot which has a linear concentration range of 50-300 ng/mL. The ICH guidelines were utilized to totally validate the presented approach. In addition the method could be efficiently incorporated in the analysis of commercial milnacipram tablets with no considerable effect on the results of the assay for milnacipran. The developed approach is characterized by its high simplicity and greenness of the procedure which make it suitable for routine analysis.

Controlling the semi-permeability of protein nanocapsules influences the cellular response to macromolecular payloads.

Nanocapsules are an excellent platform for the delivery of macromolecular payloads such as proteins, nucleic acids or polyprodrugs, since they can both protect the sensitive cargo and target its delivery to the desired site of action. However, the release of macromolecules from nanocapsules remains a challenge due to their restricted diffusion through the nanoshell compared to small molecule cargo. Here, we designed degradable protein nanocapsules with varying crosslinking densities of the nanoshell to control the release of model macromolecules. While the crosslinking did not influence the degradability of the capsules by natural proteases, it significantly affected the release profiles. Furthermore, the optimized protein nanocapsules were successfully used to deliver and effectively release a bioactive macromolecular vaccine adjuvant in vitro and, thus, can be used as an efficient platform for the design of potential nanovaccines.

In Vivo Femtosecond Laser Subsurface Cortical Microtransections Attenuate Acute Rat Focal Seizures.

Recent evidence shows that seizures propagate primarily through supragranular cortical layers. To selectively modify these circuits, we developed a new technique using tightly focused, femtosecond infrared laser pulses to make as small as ~100 µm-wide subsurface cortical incisions surrounding an epileptic focus. We use this "laser scalpel" to produce subsurface cortical incisions selectively to supragranular layers surrounding an epileptic focus in an acute rodent seizure model. Compared with sham animals, these microtransections completely blocked seizure initiation and propagation in 1/3 of all animals. In the remaining animals, seizure frequency was reduced by 2/3 and seizure propagation reduced by 1/3. In those seizures that still propagated, it was delayed and reduced in amplitude. When the recording electrode was inside the partially isolated cube and the seizure focus was on the outside, the results were even more striking. In spite of these microtransections, somatosensory responses to tail stimulation were maintained but with reduced amplitude. Our data show that just a single enclosing wall of laser cuts limited to supragranular layers led to a significant reduction in seizure initiation and propagation with preserved cortical function. Modification of this concept may be a useful treatment for human epilepsy.

Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances.

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called "edge effect"), thereby increasing usable wells to the entire plate, not just interior wells.

Multi-modal Binding of a 'Self' Peptide by HLA-B*27:04 and B*27:05 Allelic Variants, but not B*27:09 or B*27:06 Variants: Fresh Support for Some Theories Explaining Differential Disease Association.

A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169-181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (ß2 microglobulin), and in (b) peptide binding to ß2 microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27's disease association.

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A.

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine-threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [(32)P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and Vmax of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R (2) = 0.956.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

KDAC8 substrate specificity quantified by a biologically relevant, label-free deacetylation assay.

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. These modifications regulate a variety of biological processes and are reversed by the lysine deacetylase (KDAC) family of enzymes. Despite the known prevalence and importance of acetylation, the details of KDAC substrate recognition are not well understood. While several methods have been developed to monitor protein deacetylation, none are particularly suited for identifying enzyme-substrate pairs of label-free substrates across the entire family of lysine deacetylases. Here, we present a fluorescamine-based assay which is more biologically relevant than existing methods and amenable to probing substrate specificity. Using this assay, we evaluated the activity of KDAC8 and other lysine deacetylases, including a sirtuin, for several peptides derived from known acetylated proteins. KDAC8 showed clear preferences for some peptides over others, indicating that the residues immediately surrounding the acetylated lysine play an important role in substrate specificity. Steady-state kinetics suggest that the sequence surrounding the acetylated lysine affects binding affinity and catalytic rate independently. Our results provide direct evidence that potential KDAC8 substrates previously identified through cell based experiments can be directly deacetylated by KDAC8. Conversely, the data from this assay did not correlate well with predictions from previous screens for KDAC8 substrates using less biologically relevant substrates and assay conditions. Combining results from our assay with mass spectrometry-based experiments and cell-based experiments will allow the identification of specific KDAC-substrate pairs and lead to a better understanding of the biological consequences of these interactions.

Chitosan coated polylactic acid nanoparticle-mediated combinatorial delivery of cisplatin and siRNA/Plasmid DNA chemosensitizes cisplatin-resistant human ovarian cancer cells.

Development of resistance toward anticancer drugs results in ineffective therapy leading to increased mortality. Therefore, overriding resistance and restoring sensitivity to anticancer drugs will improve treatment efficacy and reduce mortality. While numerous mechanisms for drug resistance in cancer have previously been demonstrated, recent studies implicate a role for proteasome and the autophagy regulatory protein P62/SQSTM1 (P62) in contributing to drug resistance. Specifically, reduction in the expression of the ß5 subunit of the proteasome and/or enhanced P62 protein expression is known to contribute to cancer drug resistance such as cisplatin (CDDP) in ovarian cancer cells. Therefore, we hypothesized that restoration of ß5 expression and/or suppression of P62 protein expression in CDDP-resistant ovarian cancer cells will lead to restoration of sensitivity to CDDP and enhanced cell killing. To test our hypothesis we developed a biodegradable multifunctional nanoparticle (MNP) system that codelivered P62siRNA, ß5 plasmid DNA, and CDDP and tested its efficacy in CDDP resistant 2008/C13 ovarian cancer cells. MNP consisted of CDDP loaded polylactic acid nanoparticle as inner core and cationic chitosan (CS) consisting of ionically linked P62siRNA (siP62) and/or ß5 expressing plasmid DNA (pß5) as the outer layer. The MNPs were spherical in shape with a hydrodynamic diameter in the range of 280-350 nm, and demonstrated encapsulation efficiencies of 82% and 78.5% for CDDP and siRNA respectively. MNPs efficiently protected the siRNA and showed superior serum stability compared to naked siRNA as measured by gel retardation and spectrophotometry assays. The MNPs successfully delivered siP62 and pß5 to cause P62 knockdown and restoration of ß5 expression in 2008/C13 cells. Combined delivery of siP62, pß5, and CDDP using the MNPs resulted in a marked reduction in the IC50 value of CDDP in 2008/C13 cells from 125 ± 1.3 µM to 98 ± 0.6 µM (P < 0.05; 21.6% reduction) when compared to the reduction in the IC50 of CDDP observed in cells that had only siP62 delivered (IC50 = 106 ± 1.1 µM; P < 0.05; 15.2% reduction) or pß5 delivered (IC50 = 115 ± 2.8 µM; 8% reduction) via MNPs. Finally, our studies showed that the CDDP resistance index in 2008/C13 cells was reduced from 4.62 for free CDDP to 3.62 for MNP treatment. In conclusion our study results demonstrated the efficacy of our MNP in overcoming CDDP resistance in ovarian cancer cells.

Solid-phase supported profluorescent nitroxide probe for the determination of aerosol-borne reactive oxygen species.

Reactive oxygen species (ROS) and free radicals play important roles in the chemical transformation and adverse health effects of environmental aerosols. This work presents a simple and sensitive method for sampling and analysis of ROS using a packed column coated with a profluorescent nitroxide scavenger, proxyl fluorescamine (PF). Quantification was performed by extraction and analysis using HPLC with fluorescence detection. For comparison, the conventional method of collecting aerosols into dichlorofluorescin (DCFH) aqueous solution was used as a reference. The method was successfully applied to the determination of ROS in a model secondary organic aerosol (SOA) system generated by ozonolysis of nicotine, as well as in secondhand tobacco smoke (SHS). ROS concentrations between 50-565 nmol m(-3) were detected in fresh SOA and SHS samples. After SHS aging for 22 h, 13-18% of the initial ROS mass remained, suggesting the presence of persistent ROS. The new method offers better stability and reproducibility along with sensitivity comparable to that of DCFH (method detection limit of 3.2 and 1.4 nmol m(-3) of equivalent H2O2 for PF and DCFH respectively). The PF probe was stable during storage at room temperature and not reactive with ozone or NOx, whereas DCFH in the particle-collecting liquid system was strongly influenced by ozone and NOx interferences. This case study provides a good basis for employing solid-phase supported PF for field measurement of specific ROS in other combustion systems (i.e. biomass burning, candles, and diesel exhaust) and environmental aerosols.

Surface conjugation of zwitterionic polymers to inhibit cell adhesion and protein adsorption.

Non-fouling surfaces that resist non-specific protein adsorption and cell adhesion are desired for many biomedical applications such as blood-contact devices and biosensors. Therefore, surface conjugation of anti-fouling molecules has been the focus of many studies. In this study, layer-by-layer polyelectrolyte deposition was applied to create an amine-rich platform for conjugation of zwitterionic polymers. A tri-layer polyelectrolyte (TLP) coating representing poly(ethylene imine) (PEI), poly(acrylic acid)-g-azide and PEI was deposited on various polymeric substrates via layer-by-layer deposition and then crosslinked via UV irradiation. Carboxyl-terminated poly(sulfobetaine methacrylate) p(SBMA) or poly(carboxybetaine methacrylate) p(CBMA) was then conjugated onto TLP coated substrates via a carbodiimide reaction. Our results demonstrate that the zwitterionic polymers could be easily conjugated over a wide pH range except under alkaline conditions, and almost completely block protein adsorption and the attachment of L929 cells and platelets. Therefore, this method has outstanding potential in biomedical applications that require low-fouling surfaces.

On-site chemical reaction lights up protein assemblies in cells.

Here we report a fast and effective method to visualize interactive proteins across intact mammalian cells via on-site formation of fluorescence using instant reaction of non-fluorescent fluorescamine with primary amines on proteins. Without interference by fluorescence background, this fluorogenic labelling opens a way for selective identification of primary amine-rich interacting proteins, efficient mapping and real-time monitoring of their spatial distribution in assemblies/network, and fast differentiation of cellular types. Without adverse effect on biological functions, this labelling method also provides new insights to comprehend important aspects of cellular functions of organelles and their relation to health imperfections for disease diagnostics and imaging-guided therapy.

Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide.

In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates - a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization organization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α(2)ß(1) receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α(2)ß(1) receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications.

Controlling the adhesion and differentiation of mesenchymal stem cells using hyaluronic acid-based, doubly crosslinked networks.

We have created hyaluronic acid (HA)-based, cell-adhesive hydrogels that direct the initial attachment and the subsequent differentiation of human mesenchymal stem cells (MSCs) into pre-osteoblasts without osteogenic supplements. HA-based hydrogel particles (HGPs) with an average diameter of 5-6 µm containing an estimated 2.2 wt% gelatin (gHGPs) were synthesized by covalent immobilization of gelatin to HA HGPs prepared via an inverse emulsion polymerization technique. Separately, a photocrosslinkable HA macromer (HAGMA) was synthesized by chemical modification of HA with glycidyl methacrylate (GMA). Doubly crosslinked networks (DXNs) were engineered by embedding gHGPs in a secondary network established by HAGMA at a particle concentration of 2.5 wt%. The resultant composite gels, designated as HA-gHGP, have an average compressive modulus of 21 kPa, and are non-toxic to the cultured MSCs. MSCs readily attached to these gels, exhibiting an early stage of stress fiber assembly 3 h post seeding. By day 7, stellate-shaped cells with extended filopodia were found on HA-gHGP gels. Moreover, cells had migrated deep into the matrix, forming a three dimensional, branched and interconnected cell community. Conversely, MSCs on the control gels lacking gelatin moieties formed isolated spheroids with rounded cell morphology. After 28 days of culture on HA-gHGP, Type I collagen production and mineral deposition were detected in the absence of osteogenic supplements, suggesting induction of osteogenic differentiation. In contrast, cells on the control gels expressed markers for adipogenesis. Overall, the HA-gHGP composite matrix has great promise for directing the osteogenic differentiation of MSCs by providing an adaptable environment through the spatial presentation of cell-adhesive modules.

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion.

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.

Evaluation of two derivatization reagents for the determination by LC-MS/MS of ammonia in cigarette mainstream smoke.

Ammonia in cigarette mainstream smoke was quantified by LC-MS/MS after derivatization. Two different reagents, fluorescamine and dansyl chloride, were investigated, but only the latter gave stable derivatives; therefore, it was considered the most appropriate choice. Smoke samples were collected on a Cambridge filter pad followed by an impinger containing a solution of hydrochloric acid. Ammonia was then derivatized with a 18.5 mM solution of dansyl chloride in acetonitrile at 70 °C for 30 min in a vial with the internal standard, (15)ND(4)Cl. The resulting derivative was analyzed by LC-MS/MS detection with an atmospheric pressure chemical ionization (APCI) interface in the positive ionization mode using multiple-reaction monitoring (MRM). Good linearity was obtained in the concentration range of 0.02-1.65 µg/mL (r(2) ≥ 0.999), and the limit of detection (LOD) was established at 0.006 µg/mL. This method has the advantage of being sensitive, efficient, and reliable and is not hindered by interferences from the sample matrix. It should thus be considered a reference method of choice for the determination of ammonia in smoke.

Biodegradable poly(ethylene glycol) hydrogels based on a self-elimination degradation mechanism.

Two vinyl sulfone functionalized crosslinkers were developed for the purpose of preparing degradable poly(ethylene glycol) (PEG) hydrogels (EMXL and GABA-EMXL hydrogels). A self-elimination degradation mechanism in which an N-terminal residue of a glutamine is converted to pyroglutamic acid with subsequent release of diamino PEG (DAP) is proposed. The hydrogels were formed via Michael addition by mixing degradable or nondegradable crosslinkers and copolymer {4% w/v; poly[PEG-alt-poly(mercapto-succinic acid)]} at room temperature in phosphate buffer (PB, pH = 7.4). Hydrogel degradation was characterized by assessing diamino PEG release and examining morphological changes as well as the swelling and weight loss ratio under physiological conditions (37 degrees C). Degradation of EMXL and GABA-EMXL hydrogels occurred by surface erosion (confirmed by SEM). GABA-EMXL degradation was significantly faster (approximately 3-fold) than EMXL; however, the degradation of both hydrogels in mouse plasma was 12-times slower than in PBS. The slower degradation rate in plasma as compared to buffer is consistent with the presence of gamma-glutamyltransferase, gamma-glutamylcyclotransferase and/or glutaminyl cyclase (QC), which have been shown to suppress pyroglutamic acid formation. The current studies suggest that EMXL and GABA-EMXL hydrogels may have biomedical applications where 1-2 week degradation timeframes are optimal.

Ocular tolerance to a topical formulation of hyaluronic acid and chitosan-based nanoparticles.

PURPOSE: Hyaluronic acid-chitosan nanoparticles (HA-CS NPs) have the potential to serve as a reliable drug delivery system to topically treat ocular surface disorders. We evaluated the in vivo uptake by ocular structures, the acute tolerance, and possible alterations of tear film physiology in rabbits. METHODS: Fluorescent HA-CS NPs (fl-HA-CS NPs) were prepared by ionotropic gelation using fluoresceinamine-labeled hyaluronic acid and resuspended in buffer. fl-HA-CS NPs (30 microL, 0.5 mg/mL) and fluoresceinamine-HA conjugate (30 microL) were instilled into rabbit eyes every 30 minutes for 6 hours. In vivo uptake and acute tolerance were characterized 24 hours after the first instillation and compared with preinstillation measurements. Clinical signs, including tear production, lacrimal drainage system patency and reflux, and ocular surface pathology, were evaluated. RESULTS: The rabbits showed no signs of ocular discomfort or irritation after exposure to HA-CS NPs. No macroscopic alteration in ocular surface structures was observed. fl-HA-CS NPs were present inside conjunctival and corneal epithelial cells, although the distribution within the cells was different. The fl-HA-CS NPs had no significant effects on tissue morphology and functionality, tear production, or drainage. CONCLUSION: Taken together, these data demonstrate that HA-CS NPs are a safe drug carrier for ocular surface application.

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling.

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.