Galactose-Anchored Gelatin Nanoparticles for Primaquine Delivery and Improved Pharmacokinetics: A Biodegradable and Safe Approach for Effective Antiplasmodial Activity against P. falciparum 3D7 and in Vivo Hepatocyte Targeting.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

BackgroundThe objective of the current study was to assess the in vitro antiplasmodial activities of leaf, bark, flower, and the root of Pongamia pinnata against chloroquine-sensitive Plasmodium falciparum (3D7 strain), cytotoxicity against Brine shrimp larvae and THP-1 cell line. For in vivo study, the plant extract which has shown potent in vitro antimalarial activity was tested against Plasmodium berghei (ANKA strain).MethodsThe plant Pongamia pinnata was collected from the herbal garden of Acharya Nagarjuna University of Guntur district, Andhra Pradesh, India. Sequentially crude extracts of methanol (polar), chloroform (non-polar), hexane (non-polar), ethyl acetate (non-polar) and aqueous (polar) of dried leaves, bark, flowers and roots of Pongamia pinnata were prepared using Soxhlet apparatus. The extracts were screened for in vitro antimalarial activity against P. falciparum 3D7 strain. The cytotoxicity studies of crude extracts were conducted against Brine shrimp larvae and THP-1 cell line. Phytochemical analysis of the plant extracts was carried out by following the standard methods. The chemical injury to erythrocytes due to the plant extracts was checked. The in vivo study was conducted on P. berghei (ANKA) infected BALB/c albino mice by following 4-Day Suppressive, Repository, and Curative tests.ResultsOut of all the tested extracts, the methanol extract of the bark of Pongamia pinnata had shown an IC50 value of 11.67 μg/mL with potent in vitro antimalarial activity and cytotoxicity evaluation revealed that this extract was not toxic against Brine shrimp and THP-1 cells. The injury to erythrocytes analysis had not shown any morphological alterations and damage to the erythrocytes after 48 h of incubation. Because methanolic bark extract of Pongamia pinnata has shown good antimalarial activity in vitro, it was also tested in vivo. So the extract had exhibited an excellent activity against P. berghei malaria parasite while decrement of parasite counts was moderately low and dose-dependent (P < 0.05) when compared to the control groups, which shown a daily increase of parasitemia, unlike the CQ-treated groups. The highest concentration of the extract (1000 mg/kg b.wt./day) had shown 83.90, 87.47 and 94.67% of chemo-suppression during Suppressive, Repository, and Curative tests respectively which is almost nearer to the standard drug Chloroquine (5 mg/kg b.wt./day). Thus, the study has revealed that the methanolic bark extract had shown promisingly high ((P < 0.05) and dose-dependent chemo-suppression. The phytochemical screening of the crude extracts had shown the presence of alkaloids, flavonoids, triterpenes, tannins, carbohydrates, phenols, coumarins, saponins, phlobatannins and steroids.ConclusionsThe present study is useful to develop new antimalarial drugs in the scenario of the growing resistance to the existing antimalarials. Thus, additional research is needed to characterize the bioactive molecules of the extracts of Pongamia pinnata that are responsible for inhibition of malaria parasite.

The objective of the current study was to assess the in vitro antiplasmodial activities of leaf, bark, flower, and the root of Pongamia pinnata against chloroquine-sensitive Plasmodium falciparum (3D7 strain), cytotoxicity against Brine shrimp larvae and THP-1 cell line. For in vivo study, the plant extract which has shown potent in vitro antimalarial activity was tested against Plasmodium berghei (ANKA strain). The plant Pongamia pinnata was collected from the herbal garden of Acharya Nagarjuna University of Guntur district, Andhra Pradesh, India. Sequentially crude extracts of methanol (polar), chloroform (non-polar), hexane (non-polar), ethyl acetate (non-polar) and aqueous (polar) of dried leaves, bark, flowers and roots of Pongamia pinnata were prepared using Soxhlet apparatus. The extracts were screened for in vitro antimalarial activity against P. falciparum 3D7 strain. The cytotoxicity studies of crude extracts were conducted against Brine shrimp larvae and THP-1 cell line. Phytochemical analysis of the plant extracts was carried out by following the standard methods. The chemical injury to erythrocytes due to the plant extracts was checked. The in vivo study was conducted on P. berghei (ANKA) infected BALB/c albino mice by following 4-Day Suppressive, Repository, and Curative tests. Out of all the tested extracts, the methanol extract of the bark of Pongamia pinnata had shown an IC50 value of 11.67 μg/mL with potent in vitro antimalarial activity and cytotoxicity evaluation revealed that this extract was not toxic against Brine shrimp and THP-1 cells. The injury to erythrocytes analysis had not shown any morphological alterations and damage to the erythrocytes after 48 h of incubation. Because methanolic bark extract of Pongamia pinnata has shown good antimalarial activity in vitro, it was also tested in vivo. So the extract had exhibited an excellent activity against P. berghei malaria parasite while decrement of parasite counts was moderately low and dose-dependent (P < 0.05) when compared to the control groups, which shown a daily increase of parasitemia, unlike the CQ-treated groups. The highest concentration of the extract (1000 mg/kg b.wt./day) had shown 83.90, 87.47 and 94.67% of chemo-suppression during Suppressive, Repository, and Curative tests respectively which is almost nearer to the standard drug Chloroquine (5 mg/kg b.wt./day). Thus, the study has revealed that the methanolic bark extract had shown promisingly high ((P < 0.05) and dose-dependent chemo-suppression. The phytochemical screening of the crude extracts had shown the presence of alkaloids, flavonoids, triterpenes, tannins, carbohydrates, phenols, coumarins, saponins, phlobatannins and steroids. The present study is useful to develop new antimalarial drugs in the scenario of the growing resistance to the existing antimalarials. Thus, additional research is needed to characterize the bioactive molecules of the extracts of Pongamia pinnata that are responsible for inhibition of malaria parasite.

Malaria is a deadly parasitic disease, having a high rate of incidence and mortality across the world. The spread and development of resistance against chemical insecticides is one of the major problems associated with malaria treatment and control. Hence, plant based formulations may serve as an alternative source towards development of new drugs for treatment of malaria. The present study was aimed to evaluate the in vitro antiplasmodial activities of leaf, stem and flower of Calotropis gigantea against chloroquine-sensitive Plasmodium falciparum (3D7 strain) and its cytotoxicity against THP-1 cell lines. The plant extract which showed highest potency, in the in vitro antimalarial activity was further tested in vivo against P. berghei (ANKA strain) for validating its efficacy. The crude extracts of methanol, ethyl acetate and chloroform from leaves, stem and flowers of C. gigantea were prepared using Soxhlet apparatus. These extracts were screened for in vitro antimalarial activity against P. falciparum 3D7 strain. The cytotoxicity studies of crude extracts were conducted against THP-1 cell line. Phytochemical analysis of these extracts was carried out by following the standard methods. The damage to erythrocytes due to the plant extracts was tested. The in vivo study was conducted in P. berghei (ANKA) infected BALB/c albino mice by following the 4-day suppressive test. The phytochemical screening of the crude extracts showed the presence of alkaloids, flavonoids, triterpenes, tannins, carbohydrates, phenols, coumarins, saponins, phlobatannins and steroids. Out of all the extracts, the methanolic extract of leaves showed highest antimalarial activity with IC50 value of 12.17 μg/ml. In cytotoxicity evaluation, none of the crude extracts, showed cytotoxicity on THP-1 cell line. Since, methanolic leaf extract of C. gigantea showed good antimalarial activity in vitro, it was tested in vivo. In the in vivo results, the methanolic leaf extract of C. gigantea exhibited an excellent activity against P. berghei malaria parasite, wherein the decrement of parasite counts was moderately low and dose-dependent (p < 0.05) in comparison to the P. berghei infected control group, which showed a daily increase of parasitaemia unlike the chloroquine-treated group. The methanolic leaf extract of C. gigantea may act as potent alternative source for development of new medicines or drugs for the treatment of drug-resistant malaria. Thus, further research is needed to characterize the bioactive molecules of the extracts of C. gigantea that are responsible for inhibition of malaria parasite.

Formulation and characterization of spray-dried powders containing nanoparticles for aerosol delivery to the lung

Noscapine, the tubulin-binding anticancer agent, when administered orally, requires high ED(50) (300-600 mg/kg), whereas intravenous administration (10 mg/kg) results in rapid elimination of the drug with a half-life of 0.39 h. Hence, the development of long-circulating injectable nanoparticles can be an interesting option for designing a viable formulation of noscapine for anticancer activity. Noscapine-enveloped gelatin nanoparticles and poly(ethylene glycol)-grafted gelatin nanoparticles were constructed and characterized. Data indicate that smooth and spherical shaped nanoparticles of 127 ± 15 nm were engineered with maximum entrapment efficiency of 65.32 ± 3.81%. Circular dichroism confirms that nanocoacervates retained the α-helical content of gelatin in ethanol whereas acetone favored the formation of a random coil. Moreover, the Fourier transform infrared and powder X-ray diffraction pattern prevents any significant change in the noscapine-loaded gelatin nanoparticles in comparison with individual components. In-vitro release kinetic data suggest a first-order release of noscapine (85.1%) from gelatin nanoparticles with a release rate constant of 7.611×10(-3). It is to be noted that there is a 1.43-fold increase in the area under the curve up to the last sampling point for the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles over the noscapine-loaded gelatin nanoparticles and a 13.09-fold increase over noscapine. Cytotoxicity analysis of the MCF-7 cell line indicated that the IC(50) value of the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles was equivalent to 20.8 μmol/l, which was significantly (P<0.05) lower than the IC(50) value of the noscapine-loaded gelatin nanoparticles (26.3 μmol/l) and noscapine (40.5 μmol/l).Noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles can be developed as a promising therapeutic agent for the management of breast cancer.

In this study, zinc phthalocyanine (ZnPc) was loaded onto gelatin nanoparticles functionalized with polyelectrolytes (polystyrene sulfonate/polyallylamine hydrochloride) by layer-by-layer (LbL) assembly. The process yield and the encapsulation efficiency were 76.0% ± 2.5 and 86.0% ± 1.8, respectively. The functionalized photosensitive gelatin nanoparticles (FPGN) had a mean diameter of 396.5 ± 45.8 nm, narrow distribution size with a polydispersity index of 0.106. The obvious switching of zeta potential indicates successful alternating deposition of the polyanion PSS and polycation PAH directly on the gelatin nanoparticles. The in vitro drug release investigation found that the LbL deposited polyelectrolyte bilayer is very efficient to reduce the release rate and assuage the initial burst for drugs loaded in gelatin nanoparticles. The photobiological activity of FPGN was evaluated on mouse macrophage carcinoma line J774 A-1. The cells viability decreased with the increase of the light dose in the range of 1–10.0 J.cm−2. ZnPc-loaded in functionalized gelatin nanoparticles are the release systems that promise photodynamic therapy use.

This study aimed to prove the existence of anti-plasmodial activity from Tinospora crispa (T. crispa) stem extracts and to determine the IC50 values as well as the best T. crispa stem extract concentration in inhibiting the growth of P. falciparum 3D7 strain in vitro. The degree of parasitemia was observed by counting the number of infected red blood cells in Giemsa stained blood films under a light microscope with a magnification of 1000x. The Plasmodium DNA concentration was measured using flow cytometryc with Propidium Iodide (PI) staining. Analysis of fourier transform infrared (FTIR) spectrophotometer showed that the methanol extracts of T. crispa stem contained tinocrisposide compound. From the quantitative test results of Thin Layer Chromatography (TLC), it was obtained 0,22 % alkaloids that might contain compounds of berberine and palmatine. One way ANOVA statistical analysis showed that the degree of parasitemia and the concentration parasite DNA of treatment group of dose of 2,0mg/ml was significantly lower compared to the control group after 48 hours (p=0,001) and 72 hours (p=0,001) of incubation. The T. crispastem methanol extract has anti-plasmodium activity, with IC50 values between 0,27 mg/ml and 0,29mg/ml, and the effective dose to inhibit the growth of P. falciparum 3D7 strain is 2,0 mg/ml with reducing parasitemia degree by 47,12% and 56,83% after 72 hours of incubation. From this study shown the methanol extract of T. crispastem was able to reduce the parasitemia degree of P. falciparum 3D7 strain in vitro and could to be a potential candidate for anti malarias.Keywords: Antiplasmodium, methanol extract, T. crispa, Plasmodium falciparum

A small library of “half-sandwich” cyclopentadienylruthenium(II) compounds of general formula [(η5-C5R5)Ru(PPh3)(N-N)][PF6], a scaffold hitherto unfeatured in the toolbox of antiplasmodials, was screened for activity against the blood stage of CQ-sensitive 3D7-GFP, CQ-resistant Dd2 and artemisinin-resistant IPC5202 Plasmodium falciparum strains, and the liver stage of P. berghei. The best performing compounds displayed dual-stage activity, with single-digit nM IC50 values against blood stage malaria parasites, nM activity against liver stage parasites, and residual cytotoxicity against mammalian cells (HepG2, Huh7). Parasitic absorption/distribution of 7-nitrobenzoxadiazole-appended fluorescent compounds Ru4 and Ru5 was investigated by confocal fluorescence microscopy, revealing parasite-selective absorption in infected erythrocytes and nuclear accumulation of both compounds. The lead compound Ru2 impaired asexual parasite differentiation, exhibiting fast parasiticidal activity against both ring and trophozoite stages of a synchronized P. falciparum 3D7 strain. These results point to cyclopentadienylruthenium(II) complexes as a highly promising chemotype for the development of dual-stage antiplasmodials.

Polymeric nanoparticles for pulmonary protein and DNA delivery

Human malaria is caused by protozoan parasites of the genus Plasmodium and is a major contributor to global morbidity and mortality. Plasmodium falciparum is the most virulent of five Plasmodium species that infect human. As in the other species, P. falciparum develops and proliferates in the nutrient-rich anucleated erythrocyte, consuming glucose for energy and metabolizing haemoglobin as a source of amino acids. In the erythrocyte, the parasite is also able to avoid some of the host’s immune responses during infection. To support the high growth rate of the parasite during infection, glucose consumption in the infected erythrocytes increases by up to 75-fold in comparison with uninfected erythrocytes. As a consequence, a high amount of the 2-oxoaldehyde methylglyoxal, a toxic by-product of glycolysis, is spontaneously formed in the parasite. Methylglyoxal reacts with and damages nucleic acids and proteins leading to the formation of the so-called advanced glycation end-products (AGE) in the cell. Glyoxalases 1 and 2 (Glo1 and Glo2) of the ubiquitous glyoxalase system catalyze the glutathione-dependent detoxification of methylglyoxal and other 2-oxoaldehydes to non-toxic 2-hydroxycarboxylic acids. In the P. falciparum infected erythrocyte, two functional glyoxalase systems operate; one in the cytosol of the erythrocyte (hGlo1 and hGlo2) and the other in the parasite cytosol (PfGlo1 and PfcGlo2). In addition to the cytosolic enzymes, P. falciparum also encodes a functional apicoplast-targeted Glo2 enzyme (PftGlo2), and an inactive Glo1-like protein (PfGILP) that also carries an apicoplast-targeting sequence. On account of the detoxification role the glyoxalase system plays during Plasmodium infection, there has been a long-standing hypothesis that inhibition or knockout of the Plasmodium glyoxalase-encoding genes would lead to an accumulation of methylglyoxal (2-oxoaldehydes) in the host-parasite unit and result in parasite death. And so, the glyoxalase system(s) of the host-parasite unit could be targeted for antimalarial drug development. This thesis investigated the relevance of the glyoxalase system to the blood-stage parasite survival by generating clonal PFGLO1 and PFcGLO2 knockout lines of P. falciparum 3D7 strain using the CRISPR-Cas9 system. SYBR green-based growth assays of the knockout clones showed that the 3D7Δglo1 knockout clones had an increased susceptibility to the external 2-oxoaldehyde glyoxal compared with the 3D7Δcglo2 knockout clone and the 3D7 wild-type strain. Western blot analyses also supported the accumulation of selected modified proteins in 3D7Δglo1 and 3D7Δcglo2 knockout strains in comparison with the 3D7 wild-type strain. The 3D7Δglo1 and 3D7Δcglo2 knockout lines were, however, viable and showed no significant morphological or growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, resulted in increased gametocyte induction and gametocytogenesis in the knockout lines. PfGlo1 and PfcGlo2 are therefore dispensable during asexual blood-stage development and the loss of PfcGlo2 may actually contribute to the transmission of the malaria parasite. The results show that PfGlo1 and PfcGlo2 are non-essential and most likely not suitable for targeted antimalarial drug development. Several attempts to generate knockout clonal lines for the apicoplast targeted glyoxalase enzymes were unsuccessful. Transfectants resistant to both positive and negative selection were obtained in three knockout trials but were all confirmed to be false-positive transgenic parasites. The most probable explanation for this outcome is an off-target or unwanted integration of the positive selectable marker into the parasite genome. Methodological limitations, rather than the essentiality of the two enzymes, are therefore most likely the cause of the negative knockout results. The thesis also investigated the relevance of the hGlo1 enzyme to the survival of the parasite in the host-parasite unit using three tight-binding Glo1 inhibitors. Inhibitor treatments of uninfected erythrocytes showed an extremely slow inactivation of the host cell glyoxalase. Esterification did not confer improved pharmacokinetics nor increased the potency of the inhibitors. Inhibition of the erythrocyte Glo1 enzyme did not affect parasite development in the host cell, pointing to a potential dispensability of the host cell enzyme for parasite survival in the host-parasite unit. Finally, as a way of addressing the relevance of the so-called oxidative stress on parasite development, the thesis investigated the effects of the prooxidant H2O2 and the so-called antioxidants NAc, ascorbate, and DTT on the survival of P. falciparum 3D7 strain. IC50 values for the redox agents were determined for ring-stage synchronized parasites using a SYBR green-based growth assay. An IC50 value of 78 mM for H2O2 revealed the compound as a very poor prooxidant in parasite culture. The host-parasite unit appears to be very robust against challenges with H2O2 and parasite killing required extremely high concentrations with implications for host defence mechanisms. The reductants NAc, ascorbate and DTT also had antiproliferative instead of growth-promoting effects with IC50 values around 16, 4 and 0.3 mM, respectively. Taken together, the host-parasite unit appears more tolerant to high levels of H2O2, ascorbate and NAc, but is more susceptible to DTT. The inhibitory effect of the so-called antioxidants has implications for clinical trials and studies on oxidative stress.

Matrix-loaded biodegradable gelatin nanoparticles as new approach to improve drug loading and delivery

The mucosal immune system is the host's first line of defense against invasion by foreign pathogens. Gelatin nanoparticles (GNPs) are suitable carriers for the delivery of antigens via various routes of administration. In the present study, GNPs were modified with polyethyleneimine (PEI), a positively charged polymer. Then, ovalbumin (OVA) and polyinosinic:polycytidylic acid (poly(I:C)), an immunostimulant, were adsorbed onto the surface of the positively charged GNPs. We assessed whether GNPs could act as an effective mucosal vaccine that is capable of inducing both mucosal and systemic immune responses. The results showed that GNPs effectively adsorbed OVA/poly(I:C), facilitated cellular uptake by RAW 264.7 macrophage cells and murine bone marrow-derived dendritic cells (BMDCs) in vitro, and led to increased expression of the maturation markers CD80 and CD86 on BMDCs. Furthermore, GNPs induced increased secretion of proinflammatory cytokines in both RAW 264.7 and BMDCs. C57BL/6 mice that were intranasally twice-immunized with OVA/poly(I:C)-loaded GNPs produced high levels of serum OVA-specific IgG antibodies and secretory IgA in nasal and lung lavage. Spleen cells from immunized mice were collected and re-stimulated with OVA, and results showed significantly augmented production of IFN-γ, IL-4, IL-5, and IL-6 in mice that received OVA/poly(I:C)-loaded GNPs. Moreover, intranasal immunization with OVA/poly(I:C)-loaded GNPs resulted in the inhibition of EG7 tumor growth in C57BL/6 mice. Taken together, these results indicate that nasal administration of OVA/poly(I:C)-loaded GNPs elicited effective mucosal and systemic immune responses, which might be useful for further applications of antigen delivery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1228-1237, 2019.

In this study, phthalocianato[bis(dimethylaminoethanoxy)] silicon (NzPC) was loaded onto gelatin nanoparticles functionalized with polyelectrolytes (polystyrene sulfonate/polyallylamine hydrochloride) by layer-by-layer (LbL) assembly for photodynamic therapy (PDT) application in promastigote form of Leishmania amazonensis treatment. The process yield, and encapsulation efficiency were 80.0% ± 1.8 and EE = 87.0% ± 1.1, respectively. The polyelectrolytic gelatin nanoparticles (PGN) had a mean diameter of 437.4 ± 72.85 nm, narrow distribution size with a polydispersity index of 0.086. The obvious switching of zeta potential indicates successful alternating deposition of the polyanion PSS and polycation PAH directly on the gelatin nanoparticles. Photosensitizer photophysical properties were shown to be preserved after gelatin nanoparticle encapsulation. The impact of the PDT in the viability and morphology of Leishmania amazonensis promastigote in culture medium was evaluated. The PGN-NzPc presented low toxicity at the dark and the PDT was capable of decreasing the viability in more than 80% in 0.1 µmol.L−1 concentration tested. The PDT also triggered significant morphological alterations in the Leishmania promastigotes. These results reinforce the idea that the use of PGN as photosensitizers carriers is useful for PDT of Leishmania promastigotes.

Mannosylated gelatin nanoparticles enhanced inactivated PRRSV targeting dendritic cells and increased T cell immunity