A Direct Comparison of Peptide Drug Delivery Systems Based on the Use of Hybrid Calcium Phosphate/Chitosan Nanoparticles versus Unmixed Calcium Phosphate or Chitosan Nanoparticles In Vitro and In Vivo.

Nanocarriers provide a number of undeniable advantages that could improve the bioavailability of active agents for human, animal, and plant cells. In this study, we compared hybrid nanoparticles (HNPs) consisting of a calcium phosphate core coated with chitosan with unmixed calcium phosphate (CaP) and chitosan nanoparticles (CSNPs) as carriers of a model substrate, enalaprilat. This tripeptide analog is an inhibitor of angiotensin-converting enzyme and was chosen by its ability to lower intraocular pressure (IOP). In particular, we evaluated the physicochemical characteristics of the particles using dynamic light scattering (DLS) and scanning electron microscopy (SEM) and analyzed their ability to incorporate and release enalaprilat. HNPs exhibited the highest drug loading capacity and both HNPs and CSNPs demonstrated slow drug release. The comparison of the physiological effects of enalaprilat-loaded CaP particles, HNPs, and CSNPs in terms of their impact on IOP in rabbits revealed a clear advantage of hybrid nanoparticles over both inorganic and chitosan nanoparticles. These results could have important mechanistic implications for developing nano-based delivery systems for other medical, veterinary, and agricultural applications.

Chitosan-Covered Calcium Phosphate Particles Co-Loaded with Superoxide Dismutase 1 and ACE Inhibitor: Development, Characterization and Effect on Intraocular Pressure.

Improvement of the efficiency of drug penetration into the eye tissues is still an actual problem in ophthalmology. One of the most promising solutions is drug encapsulation in carriers capable of overcoming the cornea/sclera tissue barrier. Formulations on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), and an inhibitor of angiotensin-converting enzyme, enalaprilat, were prepared by simultaneous inclusion of both drugs into calcium phosphate (CaP) particles in situ with subsequent covering of the particles with 5 kDa chitosan. The formulations obtained were characterized by dynamic light scattering and scanning electron microscopy. Hybrid CaP-chitosan particles co-loaded with SOD1 and enalaprilat had a mean hydrodynamic diameter of 120-160 nm and ζ-potential +20 ± 1 mV. The percentage of the inclusion of SOD1 and enalaprilat in hybrid particles was 30% and 56%, respectively. The ability of SOD1 and enalaprilat to reduce intraocular pressure (IOP) was examined in vivo in normotensive Chinchilla rabbits. It was shown that topical instillations of SOD1/enalaprilat co-loaded hybrid particles were much more effective in decreasing IOP compared to free enzyme or free enalaprilat and even to the same particles that contained a single drug. Thus, the proposed formulations demonstrate potential as prospective therapeutic agents for the treatment of glaucoma.

Human Angiotensin I-Converting Enzyme Produced by Different Cells: Classification of the SERS Spectra with Linear Discriminant Analysis.

Angiotensin I-converting enzyme (ACE) is a peptidase widely presented in human tissues and biological fluids. ACE is a glycoprotein containing 17 potential N-glycosylation sites which can be glycosylated in different ways due to post-translational modification of the protein in different cells. For the first time, surface-enhanced Raman scattering (SERS) spectra of human ACE from lungs, mainly produced by endothelial cells, ACE from heart, produced by endothelial heart cells and miofibroblasts, and ACE from seminal fluid, produced by epithelial cells, have been compared with full assignment. The ability to separate ACEs' SERS spectra was demonstrated using the linear discriminant analysis (LDA) method with high accuracy. The intervals in the spectra with maximum contributions of the spectral features were determined and their contribution to the spectrum of each separate ACE was evaluated. Near 25 spectral features forming three intervals were enough for successful separation of the spectra of different ACEs. However, more spectral information could be obtained from analysis of 50 spectral features. Band assignment showed that several features did not correlate with band assignments to amino acids or peptides, which indicated the carbohydrate contribution to the final spectra. Analysis of SERS spectra could be beneficial for the detection of tissue-specific ACEs.

Chitosan-covered calcium phosphate particles as a drug vehicle for delivery to the eye.

Formulations on the base of an inhibitor of angiotensin-converting enzyme, enalaprilat, were prepared by the inclusion of the drug into calcium phosphate (CaP)-particles in situ, followed by the covering of the particles with 5 kDa chitosan or 72 kDa glycol chitosan and cross-linking with sodium tripolyphosphate. Physicochemical characterization of the resulted hybrid particles was conducted using dynamic light scattering, as well as scanning and transmission electron microscopy. Enalaprilat-containing particles had a mean hydrodynamic diameter 180 nm and 260 nm and ζ-potential +7 mV and +16 mV for 5 kDa and 72 kDa chitosans, respectively. In vivo studies showed that enalaprilat within particles stayed longer in the tear fluid after single instillation and caused a significantly pronounced and prolonged decrease of intraocular pressure in rabbits, especially in the case of CaP-particles, covered by glycol chitosan. Thus, such formulations demonstrate potential as prospective therapeutic agents for the treatment of eye diseases.

Nanotechnology for Topical Drug Delivery to the Anterior Segment of the Eye.

Topical drug delivery is one of the most challenging aspects of eye therapy. Eye drops are the most prevalent drug form, especially for widely distributed anterior segment eye diseases (cataracts, glaucoma, dry eye syndrome, inflammatory diseases, etc.), because they are convenient and easy to apply by patients. However, conventional drug formulations are usually characterized by short retention time in the tear film, insufficient contact with epithelium, fast elimination, and difficulties in overcoming ocular tissue barriers. Not more than 5% of the total drug dose administered in eye drops reaches the interior ocular tissues. To overcome the ocular drug delivery barriers and improve drug bioavailability, various conventional and novel drug delivery systems have been developed. Among these, nanosize carriers are the most attractive. The review is focused on the different drug carriers, such as synthetic and natural polymers, as well as inorganic carriers, with special attention to nanoparticles and nanomicelles. Studies in vitro and in vivo have demonstrated that new formulations could help to improve the bioavailability of the drugs, provide sustained drug release, enhance and prolong their therapeutic action. Promising results were obtained with drug-loaded nanoparticles included in in situ gel.

Tissue ACE phenotyping in prostate cancer.

Epithelial cells of prostate express significant level of ACE and, as a result, seminal fluid has 50-fold more ACE than plasma. The substitution of highly specialized prostate epithelial cells by tumor cells results in dramatic decrease in ACE production in prostate tissues. We performed detailed characterization of ACE status in prostate tissues from patients with benign prostate hyperplasia (BPH) and prostate cancer (PC) using new approach- ACE phenotyping, that includes evaluation of: 1) ACE activity with two substrates (HHL and ZPHL); 2) the ratio of the rates of their hydrolysis (ZPHL/HHL ratio); 3) the ratio of immunoreactive ACE protein to ACE activity; 4) the pattern of mAbs binding to different epitopes on ACE - ACE conformational fingerprint - to reveal conformational changes in prostate ACE due to prostate pathology. ACE activity dramatically decreased and the ratio of immunoreactive ACE protein to ACE activity increased in PC tissues. The catalytic parameter, ZPHL/HHL ratio, increased in prostate tissues from all patients with PC, but was did not change for most |BPH patients. Nevertheless, prostate tissues of several patients diagnosed with BPH based on histology, also demonstrated decreased ACE activity and increased immunoreactive ACE protein/ACE activity and ZPHL/HHL ratios, that could be considered as more early indicators of prostate cancer development than routine histology. Thus, ACE phenotyping of prostate biopsies has a potential to be an effective approach for early diagnostics of prostate cancer or at least for differential diagnostics of BPH and PC.

Conformational fingerprint of blood and tissue ACEs: Personalized approach.

BACKGROUND: The pattern of binding of monoclonal antibodies (mAbs) to 18 epitopes on human angiotensin I-converting enzyme (ACE)-"conformational fingerprint of ACE"-is a sensitive marker of subtle conformational changes of ACE due to mutations, different glycosylation in various cells, the presence of ACE inhibitors and specific effectors, etc. METHODOLOGY/PRINCIPAL FINDINGS: We described in detail the methodology of the conformational fingerprinting of human blood and tissue ACEs that allows detecting differences in surface topography of ACE from different tissues, as well detecting inter-individual differences. Besides, we compared the sensitivity of the detection of ACE inhibitors in the patient's plasma using conformational fingerprinting of ACE (with only 2 mAbs to ACE, 1G12 and 9B9) and already accepted kinetic assay and demonstrated that the mAbs-based assay is an order of magnitude more sensitive. This approach is also very effective in detection of known (like bilirubin and lysozyme) and still unknown ACE effectors/inhibitors which nature and set could vary in different tissues or different patients. CONCLUSIONS/SIGNIFICANCE: Phenotyping of ACE (and conformational fingerprinting of ACE as a part of this novel approach for characterization of ACE) in individuals really became informative and clinically relevant. Appreciation (and counting on) of inter-individual differences in ACE conformation and accompanying effectors make the application of this approach for future personalized medicine with ACE inhibitors more accurate. This (or similar) methodology can be applied to any enzyme/protein for which there is a number of mAbs to its different epitopes.

ACE phenotyping in Gaucher disease.

BACKGROUND: Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. METHODS: ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. RESULTS: We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10-30 kDa ACE effector which is deficient in Gaucher spleen. CONCLUSIONS: The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.

ACE phenotyping in human heart.

AIMS: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs. METHODS AND RESULTS: We performed ACE phenotyping (ACE levels, conformation and kinetic characteristics) in the human heart and compared it with that in the lung. ACE activity in heart tissues was 10-15 lower than that in lung. Various ACE effectors, LMW endogenous ACE inhibitors and HMW ACE-binding partners, were shown to be present in both heart and lung tissues. "Conformational fingerprint" of heart ACE (i.e., the pattern of 17 mAbs binding to different epitopes on the ACE surface) significantly differed from that of lung ACE, which reflects differences in the local conformations of these ACEs, likely controlled by different ACE glycosylation in these organs. Substrate specificity and pH-optima of the heart and lung ACEs also differed. Moreover, even within heart the apparent ACE activities, the local ACE conformations, and the content of ACE inhibitors differ in atria and ventricles. CONCLUSIONS: Significant differences in the local conformations and kinetic properties of heart and lung ACEs demonstrate tissue specificity of ACE and provide a structural base for the development of mAbs able to distinguish heart and lung ACEs as a potential blood test for predicting atrial fibrillation risk.

Tissue Specificity of Human Angiotensin I-Converting Enzyme.

BACKGROUND: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. METHODS/PRINCIPAL FINDINGS: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. CONCLUSIONS: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs.