This study investigated codelivery of doxorubicin (DOX) and crocetin encapsulated in PLGA nanoparticles to treat breast cancer. PLGA nanoparticles containing both DOX and crocetin (PLGA-DOX-Cro NPs) were prepared using a double emulsion/solvent evaporation method. Characterization of the nanoparticles found them to be 150-300 nm in size. In vitro studies on MCF-7 breast cancer cells showed that PLGA-DOX-Cro NPs inhibited cell growth more than DOX or crocetin alone, as measured by MTT assay and flow cytometry. Cellular uptake studies also demonstrated effective uptake of the DOX-Cro-loaded NPs by MCF
HPTLC determination of carotenoid profile in the leaf and bark samples of lor...Jing Zang
Influence of host plants on the carotenoid profile of Loranthus longiflorus leaf and bark samples collected from Casuarina equisetifolia and Ficus religiosa host trees were determined by HPTLC method. The methanol extract of L. longiflorus leaf samples obtained from C. equisetifolia host trees showed 9 compounds while it was 8 compounds in the leaf samples collected from F. religiosa host tree. Among the compounds, 5 and 3 compound in each sample, respectively, was identified as carotinoids while the others were unknown. Four compounds from each leaf samples collected from C. equisetifolia (peak no. 4- 6 & 8) and F. religiosa (peak no. 1-3 & 6) host trees showed similar Rf values (0.15, 0.19, 0.23 & 0.53, respectively). Similarly, the methanol extract of L. longiflorus bark sample collected from C. equisetifolia and F. religiosa host trees contained 8 compounds each. Of these compounds only 3 from each sample was identified as carotenoids whereas others were unknown and none of these compounds showed any similar Rf values. One compound from leaf and park samples of L. longiflorus collected from C. equisetifolia (peak no. 6 & 4) and F. religiosa (peak no. 4 & 3) showed similar Rf values (0.23 & 0.26), respectively.
ABSTRACT- Secondary metabolites or phytochemicals from plants have eminent pharmacological activities such as
anti-oxidative, anti-allergic, antibiotic, hypoglycaemic and anti-carcinogenic. These secondary metabolites protect the
cells from the damage caused by unstable molecules known as free radicals. They can inhibit oxidation of free radicals in
both human body and food system. Food industry uses both natural and synthetic antioxidants to extend shelf life of
products. But the application of synthetic antioxidant has been limited due to its carcinogenicity. Recently research is
being focused on fruit materials, which are considered rich source of antioxidant compounds. In this study the
phytoconstituents of seed extracts of two varieties of Cucumis melo L, namely Cucumis melo cantalupensis and Cucumis
melo reticulatus, were studied for their antioxidant property by DPPH free radical scavenging method. In this
investigation, chloroform, petroleum ether, acetone, aqueous and ethanolic extracts of the fruit seed were made using cold
extraction process. Phytochemical study reveals that anthroquinones, quinines, cardiac glycosides, terpenoids, phenols and
steroids were present in aqueous extract of both the samples. The total phenolic content of their seed extracts were found
to be 8.8 mg GAE/g of dry sample and 9.2 mg GAE/g of dry sample respectively. The phenolic content was found to be
linearly proportional to the antioxidant ability of the samples.
Key-words- Cucumis melo cantalupensis, Cucumis melo reticulates, DPPH, Antioxidant, Phenolic content,
Phytochemicals
Objective: The purpose of the present research investigation was to formulate sustained release (SR) formulations for
losartan potassium using 32
factorial designs. Methods: Losartan potassium is an antihypertensive agent, non-peptide
angiotensin-II receptor (type AT1) blocker, and BCS class-III agent. SR tablet formulations of losartan potassium were
formulated using variable quantities of hydroxymethyl propyl cellulose (HPMC) K100M and xanthan gum in combinations
by direct compression technique. The amount of polymers, HPMC K100M, and xanthan gum required to achieve the drug
release was selected as independent variables, X1
and X2
, respectively, whereas time required to release 10% (t10%), 50%
(t50%), 75% (t75%), and 90% (t90%) of drug from formulation was selected as dependent variables. Nine formulations were
prepared and evaluated for various pharmacopoeial tests. Results: The results reveal that all formulations were found to be
with in the pharmacopoeial limits and in vitro drug release profiles of all formulations were subjected to kinetic modeling.
The statistical parameters such as intercept, slope, and correlation coefficient were determined. Polynomial equations were
developed for dependent variables. Validity of developed polynomial equations was checked by designing two checkpoint
formulations (C1
and C2
). According to SUPAC guidelines, formulation (F4
) containing mixture of 15% HPMC K100M
and 20% xanthan gum is the most identical formulation (similarity factor f2 = 86.747, dissimilarity factor f1 = 1.760, and no
significant difference, t = 0.0477) to marketed product (LOSACAR). Conclusion: Best Formulation F4 follows the first-order,
Higuchi kinetics, and the mechanism of drug release was found to be non-Fickian diffusion anomalous transport (n = 0.825).
KEY WORDS: 32 factorial design, First-order kinetics, Hydroxymethyl propyl cellulose K100M, Losartan potassium,
Non-fickian diffusion mechanism, Sustained release tablet, Xanthan gum
Various approaches to Targeted Drug Delivery Systems (TDDS) in its formuation and evaluation in a pharmaceutical industry and research is outlined in this presentation.
COMPARSION OF ANTIOXIDANT POTENTIAL OF DIMOCARPUS LONGAN LOUR. EXTRACTS AND ...IJSIT Editor
The present study was carried out to evaluate antioxidant activity of Dimocarpus longan stems
extracts and also to investigate the main phytoconstituents in the bio-active extract. N-hexane,
dichloromethane, ethyl acetate and methanol 80% extract were tested for free radical scavenging activity on
model reaction with stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). The results showed that ethyl
acetate was the most active one as antioxidant agent and phytochemical analysis of that extract revealed the
presence of triterpenes, flavonoids, tannins and carbohydrates. The results may help to discover new
chemical classes of natural antioxidant substances that could serve as selective agents for infectious diseases.
Biopharmaceutical system , methods of permeability , generic biologics, gener...Siddhapura Pratik
Biopharmaceutical classification system, methods of permeability, generic biologics ( biosimilar drug product), clinical significance of bioequivalence studies , special concerns in bioavailability and bioequivalence studies , Generic substitution
Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Ca...Jolene1981
ABSTRACT: The use of mushrooms contributes to human nutrition by providing low lipid content of lipids and high dietary fiber content, as well as significant content of other biologically active compounds such as polysaccharides, minerals, vitamins, and polyphenolic antioxidants. This study aimed to determine the content of polyphenols and polysaccharides, as well as the cytotoxic and antioxidative properties of several medicinal mushroom preparations. The content of total phenols and flavonoids of preparations of blended mushroom extracts (Lentifom, Super Polyporin, Agarikon, Agarikon Plus, Agarikon.1, and Mykoprotect.1) was evaluated quantitatively by using ultraviolet–visible spectroscopy spectrophotometric methods. The antioxidant capacity of the preparations was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and ferric reducing/antioxidant power assays. The content of water-soluble polysaccharides was determined using a specific gravimetric method, based on ethanol precipitation. To determine cytotoxic effects of single and blended mushroom extracts, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red assays were conducted using human small cell lung cancer, lung adenocarcinoma, colon cancer, and brain astrocytoma cancer cells. The obtained results suggest that due to the significant content of beneficial polyphenolic antioxidants and soluble polysaccharides, use of these mushroom preparations is beneficial in maintaining good health, as well as in the prevention and adjuvant biotherapy of various human pathological aberrations. These results reveal that these extracts exhibit different cytotoxic effects on tumor cells originating from different tissues. In addition, the comparison of investigated blended mushroom extracts with three well-known commercial mushroom products derived from single mushroom species or single mushroom compounds shows that blended mushroom extracts exhibit significantly stronger cytotoxic effects on human tumor cell lines.
Antioxidant and-anticancer-activities-of-moringa-leavesSilentdisco Berlin
Moringa is a plantfood of high nutritional value, ecologically and economically beneficial and readily available in the countries hardest hit by the food crisis. http://miracletrees.org/ http://moringatrees.org/
HPTLC determination of carotenoid profile in the leaf and bark samples of lor...Jing Zang
Influence of host plants on the carotenoid profile of Loranthus longiflorus leaf and bark samples collected from Casuarina equisetifolia and Ficus religiosa host trees were determined by HPTLC method. The methanol extract of L. longiflorus leaf samples obtained from C. equisetifolia host trees showed 9 compounds while it was 8 compounds in the leaf samples collected from F. religiosa host tree. Among the compounds, 5 and 3 compound in each sample, respectively, was identified as carotinoids while the others were unknown. Four compounds from each leaf samples collected from C. equisetifolia (peak no. 4- 6 & 8) and F. religiosa (peak no. 1-3 & 6) host trees showed similar Rf values (0.15, 0.19, 0.23 & 0.53, respectively). Similarly, the methanol extract of L. longiflorus bark sample collected from C. equisetifolia and F. religiosa host trees contained 8 compounds each. Of these compounds only 3 from each sample was identified as carotenoids whereas others were unknown and none of these compounds showed any similar Rf values. One compound from leaf and park samples of L. longiflorus collected from C. equisetifolia (peak no. 6 & 4) and F. religiosa (peak no. 4 & 3) showed similar Rf values (0.23 & 0.26), respectively.
ABSTRACT- Secondary metabolites or phytochemicals from plants have eminent pharmacological activities such as
anti-oxidative, anti-allergic, antibiotic, hypoglycaemic and anti-carcinogenic. These secondary metabolites protect the
cells from the damage caused by unstable molecules known as free radicals. They can inhibit oxidation of free radicals in
both human body and food system. Food industry uses both natural and synthetic antioxidants to extend shelf life of
products. But the application of synthetic antioxidant has been limited due to its carcinogenicity. Recently research is
being focused on fruit materials, which are considered rich source of antioxidant compounds. In this study the
phytoconstituents of seed extracts of two varieties of Cucumis melo L, namely Cucumis melo cantalupensis and Cucumis
melo reticulatus, were studied for their antioxidant property by DPPH free radical scavenging method. In this
investigation, chloroform, petroleum ether, acetone, aqueous and ethanolic extracts of the fruit seed were made using cold
extraction process. Phytochemical study reveals that anthroquinones, quinines, cardiac glycosides, terpenoids, phenols and
steroids were present in aqueous extract of both the samples. The total phenolic content of their seed extracts were found
to be 8.8 mg GAE/g of dry sample and 9.2 mg GAE/g of dry sample respectively. The phenolic content was found to be
linearly proportional to the antioxidant ability of the samples.
Key-words- Cucumis melo cantalupensis, Cucumis melo reticulates, DPPH, Antioxidant, Phenolic content,
Phytochemicals
Objective: The purpose of the present research investigation was to formulate sustained release (SR) formulations for
losartan potassium using 32
factorial designs. Methods: Losartan potassium is an antihypertensive agent, non-peptide
angiotensin-II receptor (type AT1) blocker, and BCS class-III agent. SR tablet formulations of losartan potassium were
formulated using variable quantities of hydroxymethyl propyl cellulose (HPMC) K100M and xanthan gum in combinations
by direct compression technique. The amount of polymers, HPMC K100M, and xanthan gum required to achieve the drug
release was selected as independent variables, X1
and X2
, respectively, whereas time required to release 10% (t10%), 50%
(t50%), 75% (t75%), and 90% (t90%) of drug from formulation was selected as dependent variables. Nine formulations were
prepared and evaluated for various pharmacopoeial tests. Results: The results reveal that all formulations were found to be
with in the pharmacopoeial limits and in vitro drug release profiles of all formulations were subjected to kinetic modeling.
The statistical parameters such as intercept, slope, and correlation coefficient were determined. Polynomial equations were
developed for dependent variables. Validity of developed polynomial equations was checked by designing two checkpoint
formulations (C1
and C2
). According to SUPAC guidelines, formulation (F4
) containing mixture of 15% HPMC K100M
and 20% xanthan gum is the most identical formulation (similarity factor f2 = 86.747, dissimilarity factor f1 = 1.760, and no
significant difference, t = 0.0477) to marketed product (LOSACAR). Conclusion: Best Formulation F4 follows the first-order,
Higuchi kinetics, and the mechanism of drug release was found to be non-Fickian diffusion anomalous transport (n = 0.825).
KEY WORDS: 32 factorial design, First-order kinetics, Hydroxymethyl propyl cellulose K100M, Losartan potassium,
Non-fickian diffusion mechanism, Sustained release tablet, Xanthan gum
Various approaches to Targeted Drug Delivery Systems (TDDS) in its formuation and evaluation in a pharmaceutical industry and research is outlined in this presentation.
COMPARSION OF ANTIOXIDANT POTENTIAL OF DIMOCARPUS LONGAN LOUR. EXTRACTS AND ...IJSIT Editor
The present study was carried out to evaluate antioxidant activity of Dimocarpus longan stems
extracts and also to investigate the main phytoconstituents in the bio-active extract. N-hexane,
dichloromethane, ethyl acetate and methanol 80% extract were tested for free radical scavenging activity on
model reaction with stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). The results showed that ethyl
acetate was the most active one as antioxidant agent and phytochemical analysis of that extract revealed the
presence of triterpenes, flavonoids, tannins and carbohydrates. The results may help to discover new
chemical classes of natural antioxidant substances that could serve as selective agents for infectious diseases.
Biopharmaceutical system , methods of permeability , generic biologics, gener...Siddhapura Pratik
Biopharmaceutical classification system, methods of permeability, generic biologics ( biosimilar drug product), clinical significance of bioequivalence studies , special concerns in bioavailability and bioequivalence studies , Generic substitution
Cytotoxicity of Blended Versus Single Medicinal Mushroom Extracts on Human Ca...Jolene1981
ABSTRACT: The use of mushrooms contributes to human nutrition by providing low lipid content of lipids and high dietary fiber content, as well as significant content of other biologically active compounds such as polysaccharides, minerals, vitamins, and polyphenolic antioxidants. This study aimed to determine the content of polyphenols and polysaccharides, as well as the cytotoxic and antioxidative properties of several medicinal mushroom preparations. The content of total phenols and flavonoids of preparations of blended mushroom extracts (Lentifom, Super Polyporin, Agarikon, Agarikon Plus, Agarikon.1, and Mykoprotect.1) was evaluated quantitatively by using ultraviolet–visible spectroscopy spectrophotometric methods. The antioxidant capacity of the preparations was evaluated using the ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and ferric reducing/antioxidant power assays. The content of water-soluble polysaccharides was determined using a specific gravimetric method, based on ethanol precipitation. To determine cytotoxic effects of single and blended mushroom extracts, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red assays were conducted using human small cell lung cancer, lung adenocarcinoma, colon cancer, and brain astrocytoma cancer cells. The obtained results suggest that due to the significant content of beneficial polyphenolic antioxidants and soluble polysaccharides, use of these mushroom preparations is beneficial in maintaining good health, as well as in the prevention and adjuvant biotherapy of various human pathological aberrations. These results reveal that these extracts exhibit different cytotoxic effects on tumor cells originating from different tissues. In addition, the comparison of investigated blended mushroom extracts with three well-known commercial mushroom products derived from single mushroom species or single mushroom compounds shows that blended mushroom extracts exhibit significantly stronger cytotoxic effects on human tumor cell lines.
Antioxidant and-anticancer-activities-of-moringa-leavesSilentdisco Berlin
Moringa is a plantfood of high nutritional value, ecologically and economically beneficial and readily available in the countries hardest hit by the food crisis. http://miracletrees.org/ http://moringatrees.org/
Ultra performance liquid chromatographic method for simultaneous quantificati...Ratnakaram Venkata Nadh
Plerixafor (PLX) injections are administered to patients with cancers of lymphocytes
(non-Hodgkin’s lymphoma) and plasma cells (multiple myeloma). The main
objective of the current study was to develop a short reverse phase chromatographic
method for the simultaneous quantification of PLX and its impurities, in an injection
formulation, to reduce the time required for these quality tests. Furthermore, the
present work describes the role of nonalkyl branched nonquaternary ion pair reagent
in improving the peak shape and reducing column equilibration time. The separation
of PLX and its related substances is pH dependent (optimum pH = 2.50) and was
achieved on an octadecylsilyl (C18) column. The method was validated for its intended
purpose in accordance with the current regulatory guidelines for validation. The
proposed method can be applied for quality control, release, and stability analyses of
active pharmaceutical ingredient, PLX, as well as finished products, PLX injections
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
The aim of the present research was formulation and evaluation of anti-inflammatory drug dexibuprofen loaded chitosan-based polymeric nanoparticles (NPs) for the controlled release of dexibuprofen using different concentrations of chitosan and surfactant. Materials and Methods: Dexibuprofen, a nonsteroidal anti-inflammatory drug was encapsulated with the polymer by emulsion-droplet coalescence method (DNP1-DNP5). The NPs were characterized by drug content, particle size, zeta potential, encapsulation efficiency, and in vitro drug release. Result: DNP3 was selected as best formulation due to its ideal particle size (437.6 nm), high entrapment efficiency (88.54%), and desirable drug release (99.81 ± 0.92% at the end of 24 h). Conclusion: The present study can be concluded that the newly formulated controlled release nanoparticulate drug delivery system of dexibuprofen may be ideal and effective in the management of pain due to arthritis by allowing the drug to release continuously for 24 h.
Membrane Stabilizing And Antimicrobial Activities Of Caladium Bicolor And Che...IOSR Journals
The crude methanol extracts of whole plant of Caladium bicolor (Aiton) Vent. and leaf of Chenopodium album L. as well as their pet-ether, carbon tetrachloride, chloroform and aqueous soluble fractions were evaluated for membrane stabilizing and antimicrobial activities. At concentration 1.0 mg/ml, the carbon tetrachloride soluble fraction of C. bicolor inhibited 43.92±1.63% and 38.08±0.83 % hypotonic solution and heat induced haemolysis of RBCs, respectively. Among the extractives of C. album, the aqueous soluble fraction inhibited 47.11±0.49 % and 36.73±0.76 % hypotonic solution and heat induced haemolysis of RBCs as compared to 72.79 % and 42.12 % by acetyl salicylic acid (0.10 mg/ml), respectively. C. bicolor test samples demonstrated zone of inhibition ranging from 6.0 to 20.0 mm. The chloroform soluble fraction showed the highest zone of inhibition (20.0 mm) against Staphylococcus aureus. The test samples of C. album displayed zone of inhibition ranging from 7.0 to 13.0 mm. The highest zone of inhibition (13.0 mm) was showed by the chloroform soluble fraction against Salmonella paratyphi
Formulation and evaluation of microspheres with aceclofenacSagar Savale
Aceclofenac is an analgesic and anti-inflammatory drug that reduces fever, pain, and inflammation in rheumatoid
arthritis, osteoarthritis and ankylosing spondylitis. Aceclofenac has higher anti-inflammatory action than
conventional NSAIDs. Development of microspheres is a promising technology for controlled release and drug
targeting. Various types of microspheres such as bio-adhesive, magnetic, floating, radioactive and polymeric
microspheres are developed for various purposes. Microspheres occupied a central place in novel drug delivery, it
can targeted and localized drug delivery system. This Aceclofenac Microsphere is Prepared by using Spray drying
Technique in which release rate of drug is mainly depends on formulation composition (Eudragit RS 30 D and
Ethyl Cellulose (1:2 ratio)). Formulated microspheres were characterized for particle size, encapsulation efficiency
and In vitro studies. The optimum drug-to-polymer ratio and feed flow rate is responsible for higher percent yield,
smaller particle size and maximum encapsulation efficiency.
Background: Dennentia tripetalla (Pepper Fruit) belongs to the Annonaceae family and is abundant in Nigeria. Its
fruit in folklore medicine is used for treatment of varying ailments. While ample research evidence exists on the
plants fruit and seed, no current study exists on the toxicological profile of the plant leaves.
Methods: qualitative and quantitative phytochemicals and In vitro antioxidant assays were carried out using
standard methods. The acute toxicity study indicates that the LD50 was higher than 2000 mg/Kg body weight. Subchronic
toxicity studies was carried out using five groups of rats. Group 1 served as control, 2–5 received 100 mg/
Kg, 200 mg/Kg, 500 mg/Kg and 1000 mg/Kg body weight orally for 28 days.
Results: Post-administration biochemical analysis indicates there was increased weight in rats administered 100
mg/kg and 200 mg/kg while it reduced in the 500 mg/kg group. Significant elevations of liver function markers
were reported for 200 mg/kg and 500 mg/kg respectively. Serum and hepatic protein profiles remained unaltered.
Renal function analysis revealed elevated serum urea and creatinine for 200 and 500 mg/kg groups, elevated serum
Na+ and Ca+ and reduced serum Cl− for the 500 mg/Kg group. Elevated Kidney K+ and Ca+ levels, reduced Cl−
were significantly observed in 500 mg/Kg group. Significant rise in hepatic and renal lipid peroxidation was
observed in 200 and 500 mg/Kg groups. There were observed disarmament of the antioxidant defense systems
occasioned by rise and drop in tissue (hepatic, renal, testes, heart) Superoxide dismutase (SOD), Catalase (Cat),
Glutathione-s-transferase (GST), Glutathione peroxidase (GPx) activities in the test groups relative to control.
Histopathological examination indicated architectural aberrations at 500 and 1000 mg/kg.
Conclusions: It concluded that the plant had significant phytochemical and antioxidant properties of medical
interest and possessed toxic properties in rats when administered at a dose above 200 mg/Kg over a prolonged
period of time.
Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cellsinventionjournals
Lyciumbarbarum (Gojji berry) belongs to family Salonaceae which is found in China and Himalayan. This herb is used to prevent various diseases and in medical treatments as an alternative medicine being widely used for its antioxidant and revitalizing potential effects. In recent years, Gojji has become increasingly popular in Europe and North America as a "superfruit" and dietary supplement. The belief that herbal products do not bring any risk to health, is part of popular culture. However the term "natural" assigned to many products cannot assure no health risk. The aim of this study was to evaluate the possible genotoxic effects of aqueous extract of Lyciumbarbarum (Gojji berry) by micronucleus test and comet assay. Thirty Rattus norvegicus were divided into three equal groups: 1) experimental group, submitted to Gojji berry (200mg/kg orally); 2) positive control group (cyclophosphamide), and; 3) negative control group (distilled water). Micronucleus Tests were done by smear method of bone marrow cells performed after 48h for acute, and 72h for chronic exposure. The comet assay was performed on peripheral blood taken from the tail of each animal 4h, and 24h after intervention. Cytotoxicity was assessed by observing the DNA damage measuring the percentage of DNA in the tail (% DNA- measurement of the proportion of the total DNA present in the tail) and the tail moment (TM-tail length times the percentage of DNA in the tail), calculated by 100 nucleoids per animal and the presence of micronuclei in 2,000 polychromatic erythrocytes per animal. Analysis of variance (ANOVA) followed by Tukey test at 5% significance was used comparing the results. The data showed no significant difference in the frequency of DNA damage and the number of micronuclei between the experimental group and the negative control group. The results also suggest that the aqueous extract of Lyciumbarbarum (Gojji berry) at the dose of 200 mg/kg showed no genotoxic effect, which could, to a certain point, justifies its use.
Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cellsinventionjournals
Lyciumbarbarum (Gojji berry) belongs to family Salonaceae which is found in China and Himalayan. This herb is used to prevent various diseases and in medical treatments as an alternative medicine being widely used for its antioxidant and revitalizing potential effects. In recent years, Gojji has become increasingly popular in Europe and North America as a "superfruit" and dietary supplement. The belief that herbal products do not bring any risk to health, is part of popular culture. However the term "natural" assigned to many products cannot assure no health risk. The aim of this study was to evaluate the possible genotoxic effects of aqueous extract of Lyciumbarbarum (Gojji berry) by micronucleus test and comet assay. Thirty Rattus norvegicus were divided into three equal groups: 1) experimental group, submitted to Gojji berry (200mg/kg orally); 2) positive control group (cyclophosphamide), and; 3) negative control group (distilled water). Micronucleus Tests were done by smear method of bone marrow cells performed after 48h for acute, and 72h for chronic exposure. The comet assay was performed on peripheral blood taken from the tail of each animal 4h, and 24h after intervention. Cytotoxicity was assessed by observing the DNA damage measuring the percentage of DNA in the tail (% DNA- measurement of the proportion of the total DNA present in the tail) and the tail moment (TM-tail length times the percentage of DNA in the tail), calculated by 100 nucleoids per animal and the presence of micronuclei in 2,000 polychromatic erythrocytes per animal. Analysis of variance (ANOVA) followed by Tukey test at 5% significance was used comparing the results. The data showed no significant difference in the frequency of DNA damage and the number of micronuclei between the experimental group and the negative control group. The results also suggest that the aqueous extract of Lyciumbarbarum (Gojji berry) at the dose of 200 mg/kg showed no genotoxic effect, which could, to a certain point, justifies its use.
Genotoxicity of Goji Berry (Lyciumbarbarum) In Vivo Mammalian Cellsinventionjournals
Lyciumbarbarum (Gojji berry) belongs to family Salonaceae which is found in China and Himalayan. This herb is used to prevent various diseases and in medical treatments as an alternative medicine being widely used for its antioxidant and revitalizing potential effects. In recent years, Gojji has become increasingly popular in Europe and North America as a "superfruit" and dietary supplement. The belief that herbal products do not bring any risk to health, is part of popular culture. However the term "natural" assigned to many products cannot assure no health risk. The aim of this study was to evaluate the possible genotoxic effects of aqueous extract of Lyciumbarbarum (Gojji berry) by micronucleus test and comet assay. Thirty Rattus norvegicus were divided into three equal groups: 1) experimental group, submitted to Gojji berry (200mg/kg orally); 2) positive control group (cyclophosphamide), and; 3) negative control group (distilled water). Micronucleus Tests were done by smear method of bone marrow cells performed after 48h for acute, and 72h for chronic exposure. The comet assay was performed on peripheral blood taken from the tail of each animal 4h, and 24h after intervention. Cytotoxicity was assessed by observing the DNA damage measuring the percentage of DNA in the tail (% DNA- measurement of the proportion of the total DNA present in the tail) and the tail moment (TM-tail length times the percentage of DNA in the tail), calculated by 100 nucleoids per animal and the presence of micronuclei in 2,000 polychromatic erythrocytes per animal. Analysis of variance (ANOVA) followed by Tukey test at 5% significance was used comparing the results. The data showed no significant difference in the frequency of DNA damage and the number of micronuclei between the experimental group and the negative control group. The results also suggest that the aqueous extract of Lyciumbarbarum (Gojji berry) at the dose of 200 mg/kg showed no genotoxic effect, which could, to a certain point, justifies its use.
Evaluation of the effect of crocetin on antitumor activity of doxorubicin enc...Nanomedicine Journal (NMJ)
Objective(s): The current study reports investigation of codelivery by PLGA nanoparticles (NPs) loaded with crocetin (Cro), a natural carotenoid dicarboxylicHYPERLINK “http://en.wikipedia.org/wiki/Carboxylic_acid” acid that is found in the crocus flower, and Doxorubicin (DOX).
Materials and Methods: Double emulsion/solvent evaporation method was used for preparation of PLGA nanoparticles containing Dox and Cro. Characterizations of prepared NPs were investigated by atomic force microscopy (AFM) and dynamic light scattering analysis. In vitro Cytotoxicity of DOX and Cro loaded PLGA NPs (PLGA-DOX-Cro) on MCF-7 cell line was evaluated using MTT test. Flow cytometry experiments were implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. Furthermore the expression of caspase 3 was examined by western blot analysis.
Results: The prepared formulations had size of 150- 300 nm. Furthermore, PLGA-DOX-Cro nanoparticles inhibited MCF-7 tumor cells growth more efficiently than either DOX or Cro alone at the same concentrations, as quantified by MTT assay and flow cytometry. Studies on cellular uptake of DOX-Cro-NPs demonstrated that NPs were effectively taken up by MCF-7 tumor cells.
Conclusion: This study suggested that DOX-Cro-NPs may have promising applications in breast cancer therapy.
Effects of combination of magnesium and zinc oxide nanoparticles and heat on ...Nanomedicine Journal (NMJ)
Objective: The objective of this study was to investigate the antibacterial activities of combination of MgO and ZnO nanoparticles in the presence of heat against Escherichia coli and Staphylococcus aureus.
Materials and Methods:Bacteria were grown on either agar or broth media followed by the addition of ZnO and MgO nanoparticles. Then the combined effect of ZnO and MgO nanoparticles was investigated. Furthermore, the media containing nanoparticles were treated with mild heat and their synergistic antibacterial activity was investigated against E. coli and S. aureus in milk.
Results: The data showed that the nanoparticles used in this study had no effect on the bacteria in the agar medium. However, the results showed that ZnO and MgO nanoparticles resulted in a significant decrease in the number of E. coli (P<0.000) and S. aureus (Pd”0.05) in the broth medium. The combination of nanoparticles and mild heat exhibited a significant decrease in the number of E. coli and S. aureus indicating the synergistic effects of nanoparticles and heat.
Conclusion: Using a combination of mild heat, ZnO and MgO nanoparticles, E. coli and S. aureus can be controlled successfully in the milk. Mild heating plus ZnO and MgO nanoparticles has a synergistic effect which would reduce the need for high temperature and also the concentrations of ZnO and MgO nanoparticles required for pathogen control in minimally processed milk during maintaining.
Preparation and evaluation of electrospun nanofibers containing pectin and ti...Nanomedicine Journal (NMJ)
Objective(s):The aim of this study was to prepare electrospun nanofibers of celecoxib using combination of time-dependent polymers with pectin to achieve a colon-specific drug delivery system for celecoxib.
Materials and Methods:Formulations were produced based on two multilevel 22 full factorial designs. The independent variables were the ratio of drug:time-dependent polymer (X1) and the amount of pectin in formulations (X2). Electrospinning process was used for preparation of nanofibers. The spinning solutions were loaded in 5 mL syringes. The feeding rate was fixed by a syringe pump at 2.0 mL/h and a high voltage supply at range 10-18 kV was applied for electrospinning. Electrospun nanofibers were collected and evaluated by scanning electron microscopy and drug release in the acid and buffer with pH 6.8 with and without pectinase.
Results:Electrospun nanofibers of celecoxib with appropriate morphological properties were produced via electrospinning process. Drug release from electrospun nanofibers was very low in the acidic media; while, drug release in the simulated colonic media was the highest from formulations containing pectin.
Conclusion: Formulation F2 (containing drug:ERS with the ratio of 1:2 and 10% pectin) exhibited acceptable morphological characteristics and protection of drug in the upper GI tract and could be a good candidate as a colonic drug delivery system for celecoxib.
The combined effects of Aloe vera gel and silver nanoparticles on wound heali...Nanomedicine Journal (NMJ)
Objective(s): This study was aimed at investigating the synergy effects of Aloe vera gel and silver nanoparticles on the healing rate of the cutting wounds.
Materials and Methods: In order to determine the concentration of silver nanoparticles in Aloe vera gel, the MBC methods were applied on the most common bacteria infecting wounds, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa. The cutting wounds with Full-thickness skin were dorsally created on rats; then the rats were divided into 4 groups. The treatments groups included: mixture of Aloe vera gel and silver nanoparticles, Aloe vera gel alone and silver nanoparticles alone in addition to control groups. The treatment was carried out for 2 weeks and the size of the wound closures were measured by an image software analysis.
Results:There was no significant difference (p<0.05) in healing rate between the control and mixture group. However, there were significant differences between the silver nanoparticles and Aloe vera groups using Tukey’s analysis on the 6th, 8th and 10th days.
Conclusion:The Aloe vera gel increased the rate of wound healing whereas the silver nanoparticles had a delay effect; and when they were mixed, it was similar to the average effect of both Aloe vera gel and silver nanoparticles.
Simultaneous loading of 5-florouracil and SPIONs in HSA nanoparticles: Optimi...Nanomedicine Journal (NMJ)
Objective(s): Over the past two decades, considerable interest has been focused on utilizing biocompatible magnetic nanoparticles (MNPs) for biomedical applications. In this study, production of human serum albumin (HSA) nanoparticles using desolvation technique that were simultaneous loaded with high amounts of superparamagnetic iron oxide nanoparticles (SPIONs) and 5-flourouracil (5-FU) was investigated.
Materials and Methods: 5-FU loading (%) and SPIONs entrapment efficiency (%) were optimized using response surface methodology (RSM). The design expert software used to analyse the interactive effects of pH, 5-FU and SPIONs concentrations.
Results:The optimum conditions found to be pH of 8.2, drug concentration of 1.5 mg/ml and SPIONs concentration of 2.79 mg/ml. Under the mentioned optimum conditions, particles with the size of 111.8 nm, zeta potential of -37.1 mV, 5-FU loading of 15.8% and SPIONs entrapment efficiency of 41.1% were obtained. In vitro cumulative release of 5-FU from the nanoparticles was evaluated in phosphate buffer saline (pH 7.4, 37 °C). Results indicated that 85% of the 5-FU released during 95 h, which revealed a sustained release profile. In addition, Vibrating Sample Magnetometer (VSM) analyses confirmed the superparamagnetic properties of magnetic albumin nanoparticles manufactured under the optimum conditions.
Conclusion: According to the findings,SPIONs and 5-FU loaded HAS nanoparticles arepromising for use as novel targeted delivery system due to proper magnetic and drug release behaviours.
Antimicrobial and cytotoxicity effect of silver nanoparticle synthesized by C...Nanomedicine Journal (NMJ)
Objective(s): For the development of reliable, ecofriendly, less expensive process for the synthesis of silver nanoparticles and to evaluate the bactericidal, and cytotoxicity properties of silver nanoparticles synthesized from root extract of Croton bonplandianum, Baill.
Materials and Methods: The synthesis of silver nanoparticles by plant part of Croton bonplandianum was carried out. The formation of nanoparticles was confirmed by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), XRD and UV-Vis spectrophotometric analysis. The biochemical properties were assayed by antibacterial study, cytotoxicity assay using cancer cell line.
Results: The formation of silver nanoparticles was confirmed by UV-VIS spectroscopic analysis which showed absorbance peak at 425 nm. X-ray diffraction photograph indicated the face centered cubic structure of the synthesized AgNPs. TEM has displayed the different dimensional images of biogenic silver nanoparticles with particle size distribution ranging from 15-40 nm with an average size of 32 nm. Silver particles are spherical in shape, clustered. The EDX analysis was used to identify the elemental composition of synthesized AgNPs. Antibacterial activity of the synthesized AgNPs against three Gram positive and Gram negative bacteria strains like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa carried out showed significant zones of inhibition. The cytotoxicity study by AgNPS also showed cytotoxicity on ovarian cancer cell line PA-1 and lung epithelial cancer cell line A549.
Conclusion: The present study confirms that the AgNPs have great promise as antibacterial, and anticancer agent.
Investigation of the effect of different parameters on the phase inversion te...Nanomedicine Journal (NMJ)
Objective(s): Nanoemulsions are a kind of emulsions that can be transparent, translucent (size range 50-200 nm) or “milky” (up to 500 nm). Nanoemulsions are adequatly effective for transfer of active component through skin which facilitate the entrance of the active component . The transparent nature of the system and lack of the thickener and fluidity are among advantages of nanoemulsion.
Materials and Methods: In this study, a nanoemulsion of lemon oil in water was prepared by the phase inversion temperature (PIT) emulsification method in which the tween 40 was used as surfactant. The effect of concentration of NaCl in aqueous phase, pH and weight percent of surfactant and aqueous on the PIT and droplet size were investigated. Results: The results showed that with increasing of concentration of NaCl from 0.05 M to 1 M, PIT decrease from 72 to 50. The average droplet sizes, for 0.1, 0.5 and 1 M of NaCl in 25 ºC are 497.3, 308.1 and 189.9 nm, respectively and the polydispersity indexes are 0.348, 0.334 and 0.307, respectively.
Conclusion: Considering the characteristics of nanoemulsions such as being transparent, endurance of solution and droplet size can provide suitable reaction environment for polymerization process used in making hygienic and medical materials.
Mechanism of oxidative stress involved in the toxicity of ZnO nanoparticles a...Nanomedicine Journal (NMJ)
ZnO NPs (zinc oxide nanoparticles) has generated significant scientific interest as a novel antibacterial and anticancer agent. Since oxidative stress is a critical determinant of ZnO NPs-induced damage, it is necessary to characterize their underlying mode of action. Different structural and physicochemical properties of ZnO NPs such as particle surface, size, shape, crystal structure, chemical position, and presence of metals can lead to changes in biological activities including ROS (reactive oxygen species) production. However, there are some inconsistencies in the literature on the relation between the physicochemical features of ZnO NPs and their plausible oxidative stress mechanism. Herein, the possible oxidative stress mechanism of ZnO NPs was reviewed. This is worthy of further detailed evaluations in order to improve our understanding of vital NPs characteristics governing their toxicity. Therefore, this study focuses on the different reported oxidative stress paradigms induced by ZnO NPs including ROS generated by NPs, oxidative stress due to the NPs-cell interaction, and role of the particle dissolution in the oxidative damage. Also, this study tries to characterize and understand the multiple pathways involved in oxidative stress induced by ZnO NPs. Knowledge about different cellular signaling cascades stimulated by ZnO NPs lead to the better interpretation of the toxic influences induced by the cellular and acellular parameters. Regarding the potential benefits of toxic effects of ZnO NPs, in-depth evaluation of their toxicity mechanism and various effects of these nanoparticles would facilitate their implementation for biomedical applications.
Combined effects of PEGylation and particle size on uptake of PLGA particles ...Nanomedicine Journal (NMJ)
Abstract
Objective:
At the present study, relationship between phagocytosis of PLGA particles and combined effects of particle size and surface PEGylation was investigated.
Materials and Methods:
Microspheres and nanospheres (3500 nm and 700 nm) were prepared from three types of PLGA polymers (non-PEGylated and PEGylation percents of 9% and 15%). These particles were prepared by solvent evaporation method. All particles were labeled with FITC-Albumin. Interaction of particles with J744.A.1 mouse macrophage cells, was evaluated in the absence or presence of 7% of the serum by flowcytometry method.
Results:
The study revealed more phagocytosis of nanospheres. In the presence of the serum, PEGylated particles were phagocytosed less than non-PEGylated particles. For nanospheres, this difference was significant (P<0/05) and their uptake was affected by PEGylation degree. In the case of microsphere formulation, PEGylation did not affect the cell uptake. In the serum-free medium, the bigger particles had more cell uptake rate than smaller ones but the cell uptake rate was not influenced by PEGylation.
Conclusion:
The results indicated that in nanosized particles both size and PEgylation degree could affect the phagocytosis, but in micron sized particles just size, and not the PEGylation degree, could affect this.
Synthesis of silver nanoparticles and its synergistic effects in combination ...Nanomedicine Journal (NMJ)
Abstract
Objectives:
Biofilms are communities of bacteria attached to surfaces through an external polymeric substances matrix. In the meantime, Acinetobacterbaumannii is the predominant species related to nosocomial infections. In the present study, the effect of silver nanoparticles alone and in combination with biocides and imipenem against planktonic and biofilms of A. baumannii was assessed.
Materials and Methods:
Minimum inhibitory concentrations (MICs) of 75 planktonic isolates of A. baumannii were determined by using the microdilution method as described via clinical and laboratory standards institute (CLSI). Among all strains, 10 isolates which formed strong biofilms were selected and exposed to silver nanoparticles alone and in combination with imipenem, bismuth ethandithiol (BisEDT) and bismuth propanedithiol (BisPDT) to determine minimum biofilm inhibitory concentrations (MBIC). Subsequently, minimum biofilm eradication concentrations (MBECs) of silver nanoparticles alone and in combination with imipenem against mature biofilm of the isolates were evaluated.
Results:
Results showed that 29.3% of isolates were susceptible to silver nanoparticles and could inhibit the growth and eradicate biofilms produced by the isolates. For this reason, ∑FIC, ∑FBIC and ∑FBEC ≤ 0.05 were reported which shows synergism between silver nanoparticles and imipenem against not only planktonic cells but also inhibition and eradication of biofilms. The results of ∑FBIC >2 indicated to antagonistic impacts between silver nanoparticles and BisEDT/BisPDT against biofilms.
Conclusion:
It can be concluded that silver nanoparticles alone can inhibit biofilm formation but in combination with imipenem are more effective against A. baumannii in planktonic and biofilm forms.
Abstract
Objective(s):
Zinc oxide nanoparticles (ZNP) are increasingly used in sunscreens, biosensors, food additives and pigments. In this study the effects of ZNP on liver of rats was investigated.
Materials and Methods:
Experimental groups received 5, 50 and 300 mg/kg ZNP respectively for 14 days. Control group received only distilled water. ALT, AST and ALP were considered as biomarkers to indicate hepatotoxicity. Lipid peroxidation (MDA), SOD and GPx were detected for assessment of oxidative stress in liver tissue. Histological studies and TUNEL assay were also done.
Results:
Plasma concentration of zinc (Zn) was significantly increased in 5 mg/kg ZNP-treated rats. Liver concentration of Zn was significantly increased in the 300 mg/kg ZNP-treated animals. Weight of liver was markedly increased in both 5 and 300 mg/kg doses of ZNP. ZNP at the doses of 5 mg/kg induced a significant increase in oxidative stress through the increase in MDA content and a significant decrease in SOD and GPx enzymes activity in the liver tissue. Administration of ZNP at 5 mg/kg induced a significant elevation in plasma AST, ALT and ALP. Histological studies showed that treatment with 5 mg/kg of ZNP caused hepatocytes swelling, which was accompanied by congestion of RBC and accumulation of inflammatory cells. Apoptotic index was also significantly increased in this group. ZNP at the dose of 300 mg/kg had poor hepatotoxicity effect.
Conclusion:
It is concluded that lower doses of ZNP has more hepatotoxic effects on rats, and recommended to use it with caution if there is a hepatological problem.
Synthesis of graphene oxide-TiO2 nanocomposite as an adsorbent for the enrich...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
In our study, graphene oxide-TiO2 nanocomposite (GO/TiO2) was prepared and used for the enrichment of rutin from real samples for the first time.
Materials and Methods:
The synthesized GO/TiO2 was characterized by X-ray diffraction, scanning electron microscopy, and FT-IR spectra. The enrichment process is fast and highly efficient. The factors including contact time, pH, and amount of GO/TiO2 affecting the adsorption process were studied.
Results:
The maximum adsorption capacity for ciprofloxacin was calculated to be 59.5 mg/g according to the Langmuir adsorption isotherm. The method yielded a linear calibration curve in the concentration ranges from 15 to 200 μg/L for the rutin with regression coefficients (r2) of 0.9990. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) were found to be 8 μg/Land 28 μg/L, respectively. Both the intra-day and inter-day precisions (RSDs) were < 10% .
Conclusion:
The developed approach offered wide linear range, and good reproducibility. Owing to the diverse structures and unique characteristic, GO/TiO2 possesses great potential in the enrichment and analysis of trace rutin in real aqueous samples.
Preparation and evaluation of vitamin A nanosuspension as a novel ocular drug...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
The aim of this study was to prepare a nanosuspension formulation as a new vehicle for the improvement of the ocular delivery of vitamin A.
Material and Methods:
Formulations were designed based on full factorial design. A high pressure homogenization technique was used to produce nanosuspensions. Fifteen formulations were prepared by the use of different combinations of surfactants Tween 80, benzalkonium chloride and Pluronic and evaluated for pH, particle size, entrapment efficiency, differential scanning calorimetry (DSC), stability and drug release. Also, Draize test was used to evaluate the irritation of rabbit eye by formulations.
Results:
All formulations showed a small mean size that is well suited for ocular application. Also it was observed that the particle size decreased with increase in the amount of surfactant. Drug entrapment increased with increasing amount of surfactant. It was shown that initial and final drug release can be controlled by the ratio and the total amount of surfactants, respectively.
Conclusion:
It was concluded that the use of Tween 80 and Pluronic in the formualtions with a proper ratio does not show eye irritation and could be useful to achieve a suitable nanosuspension of vitamin A as a novel ocular delivery system.
A comparative study about toxicity of CdSe quantum dots on reproductive syste...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Medicinal benefits of quantum dots have been proved in recent years but there is little known about their toxicity especially in vivo toxicity. In order to use quantum dots in medical applications, studies ontheir in vivo toxicity is important.
Materials and Methods:
CdSe:ZnS quantum dots were injected in 10, 20, and 40 mg/kg doses to male mice10 days later, mice were sacrificed and five micron slides were prepared structural and optical properties of quantum dots were evaluated using XRD.
Results:
Histological studies of testis tissue showed high toxic effect of CdSe:ZnS in 40 mg/kg group. Histological studies of epididymis did not show any effect of quantum dots in terms of morphology and tube structure. Mean concentration of LH and testosterone and testis weight showed considerable changes in mice injected with 40 mg/kg dose of CdSe:ZnS compared to control group. However, FSH and body weight did not show any difference with control group.
Conclusion:
Although it has been reported that CdSe is highly protected from the environment by its shell, but this study showed high toxicity for CdSe:ZnS when it is used in vivo which could be suggested that shell could contribute to increased toxicity of quantum dots. Considering lack of any previous study on this subject, our study could potentially be used as an basis for further extensive studies investigating the effects of quantum dots toxicity on development of male sexual system.
Functionalization of carbon nanotubes and its application in nanomedicine: A ...Nanomedicine Journal (NMJ)
Abstract
This review focuses on the latest developments in applications of carbon nanotubes (CNTs) in medicine. A brief history of CNTs and a general introduction to the field are presented.
Then, surface modification of CNTs that makes them ideal for use in medical applications is highlighted. Examples of common applications, including cell penetration, drug delivery, gene delivery and imaging, are given. At the same time, there are concerns about their possible adverse effects on human health, since there is evidence that exposure to CNTs induces toxic effects in experimental models. However, CNTs are not a single substance but a growing family of different materials possibly eliciting different biological responses. As a consequence, the hazards associated with the exposure of humans to the different forms of CNTs may be different. Understanding the structure–toxicity relationships would help towards the assessment of the risk related to these materials. Finally, toxicity of CNTs, are discussed. This review article overviews the most recent applications of CNTs in Nanomedicine, covering the period from 1991 to early 2015.
The role of surface charge of ISCOMATRIX nanoparticles on the type of immune ...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice.
Materials and Methods:
Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice.
Results:
The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups.
Conclusion:
The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.
Abstract
In the last decade, developments in nanotechnology have provided a new field in medicine called “Nanomedicine”. Nanomedicine has provided new tools for photodynamic therapy. Quantum dots (QDs) are approximately spherical nanoparticles that have attracted broad attention and have been used in nanomedicine applications. QDs have high molar extinction coefficients and photoluminescence quantum yield, narrow emission spectra, broad absorption, large effective stokes shifts. QDs are more photostable and resistant to metabolic degradation. These photosensitizing properties can be used as photosensitizers for Photodynamic Therapy (PDT). PDT has been recommended for its unique characteristic, such as low side effect and more efficiency. Therefore, nanomedicine leads a promising future for targeted therapy in cancer tumor. Furthermore, QDs have recently been applied in PDT, which will be addressed in this review letter. Also this review letter evaluates key aspects of nano-particulate design and engineering, including the advantage of the nanometer scale size range, biological behavior, and safety profile.
Preparation of protein-loaded PLGA-PVP blend nanoparticles by nanoprecipitati...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Despite of wide range applications of polymeric nanoparticles in protein delivery, there are some problems for the field of protein entrapment, initial burst and controlled release profile.
Materials and Methods:
In this study, we investigated the influence of some changes in PLGA nanoparticles formulation to improve the initial and controlled release profile. Selected parameters were: pluronic F127, polysorbate 80 as surfactant, pH of inner aqueous phase, L/G ratio of PLGA polymer, volume of inner aqueous phase and addition of polyvinylpyrrolidone as an excipient. FITC-HSA was used as a model hydrophilic drug. The nanoparticles were prepared by nanoprecipitation.
Results:
Initial release of FITC-HSA from PLGA-tween 80 nanoparticles (opt-4, 61%) was faster than control (PLGA-pluronic) after 2.30 h of incubation. Results showed that decrease in pH of inner aqueous phase to pI of protein can decrease IBR but the release profile of protein is the same as control. Release profile with three phases including a) initial burst b) plateau and c) final release phase was observed when we changed volume of inner aqueous phase and L/G ratio in formulation. Co-entrapment of HSA with PVP and pluronic reduced the IBR and controlled release profile in opt-19. Encapsulation efficiency was more than 97% and nanoparticles size and zeta potentials were mono-modal and -18.99 mV, respectively.
Conclusion:
In this research, we optimized a process for preparation of PLGA-PVP-pluronic nanoparticles of diameter less than 300 nm using nanoprecipitation method. This formulation showed a decreased initial burst and long lasting controlled release profile for FITC-HSA as a model drug for proteins.
Abstract
Objective(s):
Abdominal adhesions are one of the most important problems, occurring after intra-abdominal surgery in more than 90% of cases. This condition is the leading cause of bowel obstruction, infertility, and abdominal/pelvic pain. Gold nanoparticles (GNPs) have been shown to be non-toxic and exhibit anti-inflammatory, anti-angiogenic and antioxidant activities. The purpose of this study was to determine the effect of intraperitoneal lavage with GNP solutions on the development of postoperative peritoneal adhesion (PPA).
Materials and Methods:
In the current experimental study, thirty-five male Wistar rats were randomly assigned to seven groups of five rats. After a standardized peritoneal injury, GNP solutions in different concentrations (1, 2.5, 5, 10, 50 and 100 ng/ml) were locally administered through nebulization; normal saline (NS) was administered to the control group. Two weeks later, the rats were sacrificed and cecum and peritoneal samples were harvested for histopathological assessment. Blood samples were obtained to determine serum concentrations of inflammatory biomarkers including tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and vascular endothelial growth factor (VEGF).
Results:
The rats treated with GNPs had significantly lower microscopic and macroscopic peritoneal adhesion scores, compared to the control group (P<0.05). Score 5 of macroscopic adhesions was reported in all the rats of the control group, unlike the GNP groups. Furthermore, microscopic adhesions were reported with all rats in the control group, unlike the GNP groups (reported in 0 out of 5 rats in all GNP groups). In addition, serum levels of IL-1β, TNF-α and VEGF underwent no significant changes.
Conclusion:
Compared to the control group, GNPs decreased the severity of peritoneal adhesions, although they did not alter TNF-α, IL-1β or VEGF serum levels.
Effect of gold nanoparticles on postoperative peritoneal adhesions in rats
Nmj61931451593800
1. Nanomed. J., 3(1): 23-34, Winter 2016
23
Evaluation of the effect of crocetin on antitumor activity of doxorubicin
encapsulated in PLGA nanoparticles
1
F. Alebooye Langroodi; 1
Z. Hafezi Ghahestani; 2
M. Alibolandi; 3
M. Ebrahimian; 4*
M. Hashemi
1
Department of Biology, School of Science, Payame Noor University, Mashhad, Iran
2
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad,
Iran
3
Biotechnology Section, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
4
Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad,Iran
ABSTRACT
Objective(s): The current study reports investigation of codelivery by PLGA nanoparticles (NPs) loaded with crocetin
(Cro), a natural carotenoid dicarboxylicHYPERLINK “http://en.wikipedia.org/wiki/Carboxylic_acid” acid that is found
in the crocus flower, and Doxorubicin (DOX).
Materials and Methods: Double emulsion/solvent evaporation method was used for preparation of PLGA nanoparticles
containing Dox and Cro. Characterizations of prepared NPs were investigated by atomic force microscopy (AFM) and
dynamic light scattering analysis. In vitro Cytotoxicity of DOX and Cro loaded PLGA NPs (PLGA-DOX-Cro) on
MCF-7 cell line was evaluated using MTT test. Flow cytometry experiments were implemented to distinguish cells
undergoing apoptosis from those undergoing necrosis. Furthermore the expression of caspase 3 was examined by
western blot analysis.
Results: The prepared formulations had size of 150- 300 nm. Furthermore, PLGA-DOX-Cro nanoparticles inhibited
MCF-7 tumor cells growth more efficiently than either DOX or Cro alone at the same concentrations, as quantified by
MTT assay and flow cytometry. Studies on cellular uptake of DOX-Cro-NPs demonstrated that NPs were effectively
taken up by MCF-7 tumor cells.
Conclusion: This study suggested that DOX-Cro-NPs may have promising applications in breast cancer therapy.
Keywords: Breast cancer, Chemotherapy, Crocetin; Doxorubicin, PLGA
Nanomed. J., 3(1): 23-34, Winter 2016
DOI: 10.7508/nmj.2016.01.003
*Corresponding Author Email: hashemim@mums.ac.ir
Tel: (+98) 51-37112471
Note. This manuscript was submitted on July 28, 2015;
approved on October 2, 2015
Received; 28 July 2015 Accepted; 2 October 2015
INTRODUCTION
To improve therapeutic efficacy and reduce toxicity
and frequency of cancer drug administration, various
nanocarriers have been developed. Over the past few
decades, there has been an increasing interest in the
potential use of polymeric nanoparticles (NPs) as
delivery vehicles for chemotherapeutic drugs and the
studies have demonstrated that these nanocarriers can
signiûcantly enhance the anti-tumor efficiency of
various chemotherapeutic drugs [1-5].
Drug delivery systems which enable codelivery of
drugs with different physiochemical properties to the
same tumor cells in vitro and in vivo have been proposed
to reduce the dosage of each drug and to achieve the
synergistic effect in cancer chemotherapy [6, 7].
Recently, many multifunctional delivery systems have
been designed for codelivery of different agents,
including micelles [8-10], liposomes [11], and inorganic
nanoparticles [12].
Although many efforts have been made on a single
carrier for two or more therapeutic agents, such as
chemicals, natural products, siRNA, plasmid DNA or
peptide [13-15], codelivery of chemotherapeutic drugs
with different water solubility characteristics is not easy
to handle.Nevertheless, for the preparation of
multifunctional drug delivery systems, PLGA (poly- D,
L lactide-co glycolide) is the ideal candidate. PLGA-
based nanoparticles have gained great interest in
diagnostics and treatment applications such as
ORIGINAL RESEARCH PAPER
2. Nanomed. J., 3(1): 23-34, Winter 2016
24
F. Alebooye Langroodi et al.
preparation of sustained drug release systems [16]. On
the other hand, PLGA have been approved by FDA for
preparation of drug delivery systems.
In comparison with other carriers such as liposome,
PLGA NPs is a sophisticated vehicle for drug delivery
because of its unique characteristics comprising
biocompatibility, bioavailability, high drug-loading
capacity, stability and sustained drug release [17-20].
There are various PLGA based formulations in clinic
including Trelstar Depot for prostate cancer,
Sandostatin LAR for acromegaly and Nutropin Depot
for growth deficiencies [21]. Numerous drugs using
PLGA as carrier is also at thedifferent pre-clinical stage.
Doxorubicin (DOX) is the chemotherapeutic drug which
extensively used in clinical application, due to their
good anti-tumor efficiency against various tumors.
Doxorubicin exhibits limited efficacy in extending time
of therapy due to drug resistance and severe toxic side
effects [22-26]. Some cancer patients use natural
products derived from plants or nutrients, concurrently
with routine chemotherapeutic regime and/or radiation
therapy. Demand for new drugs has encouraged studies
investigating feasible anti-cancer compounds in fruits,
vegetables, herbs and spices. In this regards, saffron
is a spice which derivate from the plant Crocus sativus
L., has been implemented as a traditional ancient
medicine against various disease including cancer [27].
It was previously reported that crocetin, an important
active carotenoid of saffron, has significant anti-tumour
effect in vitro and in vivo [28].
Crocetin inhibits the proliferation of cancer cells by
preventing nucleic acid synthesis, enhancing apoptosis
and interrupting growth factor signaling pathways
[29].
Therefore, our strategy is to design biodegradable
PLGA nanoparticles loaded with DOX and crocetin in
order to improve therapeutic efficacy and reduce
frequency of drug administration. In this study, MCF-
7 was utilized as the in vitro tumor model to investigate
the enhancement effect of DOX by crocetin.
MATERIALS AND METHODS
Materials
Poly(d,l-lactic-co-glycolic acid) (PLGA) (Average
Mw
: 7000-17000; lactic acid : glycolic acid = 50:50) and
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) were obtained from Sigma Aldrich
(Germany). Doxorubicin hydrochloride (DOX) was
purchased from Euroasia Co., Ltd. (Delhi, India).
Crocetin was extracted from plant Crocus sativus L.
based on the method represented in the Iran Patent
No.84459.
Roswell Park Memorial Institute (RPMI) 1640 medium,
foetal bovine serum (FBS) and trypsin were purchased
from GIBCO (Germany). Polyvinyl alcohol (PVA, 87-
89% hydrolyzed, averageMw=88,000-97,000) and other
solvent and chemical reagents were procured from
Merck (Germany) without further purification.
Preparation of PLGA NPs loaded with DOX
PLGAnanoparticlesloaded with DOXwere prepared
using a water-in-oil-in-water (W/O/W) double
emulsification and solvent evaporation technique [30].
Different parameters such as concentration of PVA as
stabilizer(2and 5%),drug/polymerratios(1:10 and 1:20),
organic solutions (Dichloromethane and
Dichloromethane: acetone) and route of solvent
removal (3 hours and overnight stirring) were
considered. 25 mg PLGA was dissolved in 1 ml
dichloromethane:acetone with 4:1 ratios, mixed with 0.2
mL deionized water containing 1.25 mg doxorubicin
(drug/polymer ratio 1:20).
The mixture was sonicated in ice bath (pulse on 1 s,
pulse off 1 s, amplitude: 90%) for 1 min using a probe
sonicator (Fisons Instruments Ltd, Crawley, U.K).
The primary water-in-oil (W/O) emulsion was added to
4 mL of ice-cold 2% or 5% polyvinyl alcohol (PVA)
solution (w/v) and emulsified for 10 min using a probe
sonicator (Fisons Instruments Ltd, Crawley, U.K). The
final emulsion was drop wise added to 5 ml of PVA
0.1% for diffusion under rapidly stirring. The organic
solvent of emulsion was then removed under different
conditions (overnight stirring, 3 hours stirring or
vacuuming overnight).
The NPs were collected by centrifugation at 14000 rpm
for 25 min and washed three times with deionized water.
The final NPs were obtained as light orange powders
after lyophilisation. The blank NPs (without drugs) were
prepared by the same method.
Preparation of PLGA NPs loaded with DOX-Cro
25 mg PLGA was dissolved in dichloromethane:
acetone with 4:1 ratio, mixed with 0.2 mL deionized
water containing 1.25 mg doxorubicin with addition of
crocetin solution (1.25 mg in pyridine).
The mixture was sonicated in ice bath (pulse on 1 s,
pulse off 1 s, amplitude: 90%) for 1 min using a probe
sonicator (Fisons Instruments Ltd, Crawley, U.K). The
3. Nanomed. J., 3(1): 23-34, Winter 2016
25
primary water-in-oil (W/O) emulsion was added to 4
mL of ice-cold 5% polyvinyl alcohol (PVA) solution
(w/v) and emulsified for 10 min using a probe sonicator
(Fisons Instruments Ltd, Crawley, U.K).
The organic solvent of emulsion was then removed
overnight stirring. The NPs were collected by
centrifugation at 14000 rpmfor 25 min and washed three
times with deionized water in order to remove pyridine
and PVAresidues. The final NPs were obtained as light
orange powders after lyophilisation.
Surface morphology of PLGA NPs
The surface morphology of PLGA NPs was studied
by atomic force microscopy (AFM). An atomic force
microscope (AFM, model:Nano Wizard II NanoScience
AFM, JPK InstrumentsInc., Germany) was implemented
to investigate the structures of the prepared NPs.
The sample forAFM analysis was prepared as follows:
Microscope glass slide was washed with acetone and
ethanol, rinsed with ultrapure water and dried. After
cleaning, the prepared PLGA NPs were diluted in
deionized water (0.1 mg/mL), dispensed onto glass
slide and dried at room temperature for 24 h [31].
The measurements were performed in the tapping mode
and dehydrated state in the air, and ACT cantilevers
(JPK Instruments Inc., Germany) were used.
Particle size analyzer
The measurement of NPs size and size distribution
was carried out by dynamic light scattering. Briefly 1
mg lyophilized NPs was dissolved in 1000 µl of
deionised water and then after sonication, analyzed
with zetasizer (NANO-ZS, Malvern, UK) at a scattering
angle of 90p equipped with a 4 mW He-Ne laser
operating at 633 nm using back-scattering detection.
All measurements were performed in triplicate at 25°C
[32].
Drug loading and encapsulation efficiency
The amount of DOX in NPs was analyzed using a
Jasco FP-6200 spectrofluorometer (Tokyo, Japan) at
λexcitatin
=470 nm, λemission
= 590 nm by dissolving 1 mg
NPs in 1 mL dimethylsulfoxide (DMSO) and employing
the calibration curve of 0–100 μg/mL DOX in the same
solvent.
The amount of crocetin in NPs was analyzed using a
Shimatzu UV–visible spectrophotometer (Tokyo, Japan)
at 430 nm using the calibration curve of 2-20 μg/mL
crocetin in the same solvent.
Each experiment was conducted in triplicate.
The encapsulation efficiency (EE%) and loading
content (LC%) of PLGA NPs loaded with crocetin and/
or DOX were calculated by the following equations
EE (%) =Amount of drug in NPs/amount of drug used
for encapsulation×100
LC (%)= Mass of drug in NPs/ Mass of NPs ×100
Release study
NPs suspension were prepared by dissolving 10
mg of freeze-dried formulation powder in 10 mL
Phosphate buffer saline (PBS 0.1 M, pH 7.4) containing
0.1% v/v of Tween 80 (to maintain a sink condition)
[33]. The NP suspension was equally divided in ten
tubes containing 1 mL and kept in a shaker incubator
set at 90 rpm and 37p C. At specific intervals these
tubes were taken out from shaker and centrifuged at
14000 rpm for 10 minutes. The supernatant was
lyophilized and dissolved in 1 mL DMSO. The amount
of DOX and Cro in the sample was determined as
described previously. Each drug release experiment
was repeated three times.
MTT assay
MCF-7 cells were cultured in the RPMI medium,
supplemented with 10% (v/v) heat-inactivated foetal
bovine serum (FBS), 1% penicillin-streptomycin at 37
°C and 5% CO2
.
MCF-7 cells were seeded into 96-well plates at 1×104
and incubated for 24 hours. DOX and Cro with different
concentrations (DOX: 0.5, 1, 2, 4, 8 µM, Cro: 1, 2, 4, 8,
18 µM) were prepared in RPMI containing 10% FBS.
Blank PLGA NPs, DOX, DOX+Crocetin, , PLGA-Dox
NPs, PLGA-Dox NPs+ free Crocetin, PLGA-Dox-Cro
NPs (equivalent aforementioned concentrations of
DOX and Cro either in solution or in PLGA NPs
formulation) were also dispersed in RPMI containing
10%FBS.
The prepared drug concentration was added to each
well, followed by incubation of the cells for 48 h.
20 µL MTT (5 mg/mL in PBS) solution was then added
to each well. After 4 h, MTT solution was aspirated
from the wells using vacuum and 100 µL DMSO was
added to each well. The absorbance at 570 nm with a
reference wavelength at 630 nm, was measured by a
Infinite® 200 PRO multimode microplate reader (Tecan
Group Ltd. Männedorf, Switzerland).
Apoptosis assay
MCF-7 cells were exposed to DOX, PLGA NPs
containing equivalent amount of doxorubicin or
4. Nanomed. J., 3(1): 23-34, Winter 2016
26
the effect of crocetin on antitumor activity of doxorubicin-PLGA
doxorubicin along with crocetin for 48 h at 37°C and
then detached and collected.
The untreated and treated MCF-7 cells were washed
twice with phosphate buffer, Propidium iodide was then
added to a final concentration 50 µg/mL which can
bind to double stranded DNA by intercalating between
base pairs. Then the mixture was stored in the dark at
4°C for 2 h.
At final stage, the fluorescence of 10,000 fixed cells
which stained with propidium iodide was assayed on a
FACScaliber (Bector Dickinson, USA) using FL-2
channel [34]. The number of apoptotic cells was
analyzed using the WinDMI 2.9 software.
Western blot analysis
The untreated and treated cells with different
formulations or free drugs were harvested, lysed, and
their protein concentrations were quantiûed by the
Bradford assay [35].
The equal amounts of protein from each sample were
separated by electrophoresis on a 12% denaturing SDS
gel. Proteins were transferred onto 0.45 µm pore size
PVDF membrane (BioRad, USA). Detection was
conducted by immuno-staining using 1:1000 dilution
speciûc primary antibodies of caspase-3 (9665s-Cell
Signaling) and horseradish peroxidase-conjugated anti-
IgG antibody (1:3000 dilution) (7074s-Cell Signaling).
The protein bands were visualized by the enhanced
chemiluminescence (ECL) assay according to the
manufacturer’s instructions.
Cellular uptake
MCF-7 cells were seeded in 12-well plates at 8 × 104
cells per well and cultured for 24 h.
The cells were then incubated at 37 °C for 2h, 24h and
48h with free drugs or prepared formulations
(equivalent to 1 µM doxorubicin) in RPMI containing
10%FBS.
Then, the culture medium was removed; the cells were
then washed by PBS and were trypsinized and
centrifuged at 1500 rpm for 10 min.
The supernatant were removed, and the pellet was
washed three times with cold PBS. At the final stage,
collected cells were suspended in 1 mL cold PBS and
the intensity of the doxorubicin ûuorescence in the
cells was determined using a FACScaliber (Bector
Dickinson, USA) equipped with a 488 laser in the FL1
channel. The data were analysed using the WinDMI
2.9 analysis software.
Statistical analysis
The results were reported as means ± SD (n e” 3).
Data were analysed by one-way analysis of variance
(ANOVA). A probability value of less than 0.05 was
considered signiûcant.
RESULTSANDDISCUSSION
Preparation of DOX or DOX/ Cro loaded PLGA NPs
In order to overcome the undesirable side effects
of drugs, synergistic combination of two or more
drugs is a versatile strategy [3].
In comparison with a single drug delivery system,
codelivery of multiple drugs has numerous advantages
including reducing administration dosage of each
drug because of enhancement effect, suppressed drug
resistance, reducing undesirable toxicity and
accessing context-speciûc multiple targets.
Therefore, it is crucial to codeliver two or more drugs
through a nanocarrier and convey them to the same
cancer cell coincidentally [2, 5].
In the development of the codelivery systems,
encapsulating drugs with different water solubility
properties is a serious challenge.
The PLGA nanoparticles can be produced by various
techniques. Usually emulsification solvent
evaporation technique is used for PLGA NPs
preparation because of its simplicity and high drug
loading capacity.
The single emulsion method is a very useful method
for encapsulation of hydrophobic drugs and leads to
very low encapsulation efficiency of hydrophilic
drugs.
The double emulsion method is a versatile technique
for encapsulating both hydrophilic and hydrophobic
drugs with high efficiency [36-38].
This technique allows preparation of NPs which
encapsulated both hydrophobic and hydrophilic
cargos.
Therefore, in this study, we developed a convenient
W/O/W method for development of doxorubicin
(hydrophilic drug) and crocetin (hydrophobic drug)
co-encapsulated PLGA NPs.
Table 1 summarized the average particles size of Dox-
loaded PLGA NPs prepared under different conditions.
Four quantitative independent variables consisting
of organic phase, concentration of PVA, concentration
ratio of polymer/drug, route of solvent removal and
dependent variables were diameter and polydispersity
of NPs.
5. Nanomed. J., 3(1): 23-34, Winter 2016
27
In PLGA formulations, the optimal condition was as
follows: PVA 5%, 1:20 ratio of drug to polymer,
dichloromethane:acetone (4:1) as organic solvent and
overnight stirring for solvent removal. The particle
size of the nanoparticles which prepared under
aforementioned condition was about 178 nm. Obtained
results demonstrate that the concentration of surfactant
(PVA) and route of solvent removal was crucial for
preparation of nanosized drug loaded particles
(<300nm).
Both doxorubicin and crocetin were successfully
entrapped in the PLGA NPs (F6 formulation) prepared
by double emulsion method.
The drug loading efficiency was about 54% and 65%
for DOX and Cro respectively. On the other hand, the
loading content was also calculated to be 2.43% and
2.94% for DOX and Cro respectively. It seemed that
the loading efficiency of Cro is higher than that of DOX.
This was owing to that Cro is a hydrophobic compound,
while DOX is a hydrophilic drug and may leak out to
the external water phase during the emulsification. The
real molar ratio of DOX/Cro in formulation was fixed.
Morphology of NPs
The morphology of PLGA-DOX-Cro NPs (F6) was
examined by AFM (Fig. 1). It was revealed that the
prepared NPs were homogeneously distributed as
individual well-deûned spherical particles around a size
of200-300 nm.
In vitro drug release
The controllable release behaviour of drugs can be
achieved by using polymeric nanocarriers for
simultaneous encapsulation of two or more drugs in a
vehicle.
Then prolonged release time, enhancement effects of
encapsulated cargos and reduced administration
frequency could be achieved for crocetin-doxorubicin
loaded PLGA NPs.
The in vitro drug release experiment of F6 formulation
has been conducted in a PBS solution at pH 7.4. As
illustrated in Fig. 2, DOX released a little faster than
Cro in the first few hours, and the release rates were
similar after 20 hours.
This might be caused by some of the hydrophilic DOX
was adhered to the surface of the PLGA NPs. Therefore,
DOX released faster when the NPs were suspended in
PBS.Approximately 80% of the drugs (DOX and Cro)
were accumulatively released within 48 hours.
Doxorubicin and crocetin releases from PLGANPs were
nearly linear for the first 5 hours, with 60% and 40%
DOX and Cro release respectively. Drug loaded PLGA
nanoparticles often display burst release, which can
be attributed to unencapsulated drug localized on the
surface or near the surface of the nanoparticles [39,
40]. Also, the presence of suspended (undissolved)
drug in the polymer matrix probably contributes to the
linear drug release as reported previously [41]. It would
appear that the suspended drug provides a constant
driving force for diffusion of cargos (doxorubicin and
crocetin) out of the nanoparticles.
In the present study, release profile clearly suggests a
diffusion mode of release for both drugs in first 5 hours
and then erosion being the main mechanism of release
until complete release of drugs up to 48 hours.
This type of release pattern for DOX and Cro could be
advantageous in cancer chemotherapy. The initial burst
release over 5 h provides a primary dose, and the
remaining dose is released during 48 hrs.
Such a controlled release system by which the drug
will be available to the tumour tissue in an instantaneous
manner is of significant importance.
Combinational effects of DOX and Cro on MCF-7
cell lines
Crocetin is a natural carotenoid and is one of the
major active compounds of saffron. Crocetin consist
of 20 carbon atoms including double bonds and a
carboxylic acid group at each end of the chain (Fig. 3).
Previously various pharmacological effects of crocetin
were reported; comprising its antioxidant, anti-
inûammatory, and anti-tumor effects [42, 43].
Recently, crocetin has attracted more attention as a
potent natural product with anti-tumor activity.
Dehr et al. [44] demonstrated that crocetin can inhibit
tumor growth in pancreatic cancer xenograft mouse
model. Furthermore, crocetin prevents 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced skin
tumors in mice[45] and shows protective effects against
benzo (a)pyrene induced lung cancer[46].
Several studies have demonstrated that crocetin or
crocetin containing combination regimens are very
effective in the treatment of cancer.
Zhong et al. [47] demonstrated that crocetin caused
cytotoxicity in cancerous cells by enhancing apoptosis
in a time-dependent manner. Also they proved that the
crocetin has synergistic effect on the cytotoxicity
induced by vincristine.
6. Nanomed. J., 3(1): 23-34, Winter 2016
28
F. Alebooye Langroodi et al.
Li et al. [48] also evaluated the cytotoxicity of corcetin
in colon cancer cell line and approved p-53-independent
cellular toxicity of cocetin. Altogether these reports
verified the potency of crocetin as a versatile anti-cancer
agent. Herein, in order to investigate the enhancement
antitumor effect of DOX in the PLGA NPs by crocetin,
the cytotoxicity of PLGA-DOX NPs, PLGA-DOX-Cro
NPs,PLGA-DOXNPs+freeCro,freeDOXandfreeDOX+
free Cro in MCF-7 cell line was evaluated. The Dox and
Cro concentrations in these formulations were 0.5-10
µM and 1-16 µM respectively. In our study, Cro in 1-16
µM concentrations showed no cytotoxicity on MCF-7
cell line (Fig. 4A). Then, allprepared formulations contain
nontoxic concentrations of Cro along with DOX.
T a b le 1 . P re p a ra tio n o f d ru g lo a d e d P L G A N P s u sin g d o u b le e m u lsio n m e th o d u n d e r d iffe re n t c o n d itio n s
D O X e n c a p su la tio n
P D IS iz e (n m )S o lv e n t re m o v a lP V AD C M (µ l):A c e to n e (µ l)D O X: P o ly m e rF o rm u la tio n
0 .12 3 0S tirrin g o v e rn ig h t
5%
4 m l
1 0 0 0 :01 :1 0F1
0 .12 2 2 .9S tirrin g o v e rn ig h t
5%
4 m l
8 0 0 :2 0 0
1 :1 0F2
0 .43 6 43 h o u rs stirrin g
5%
4 m l
8 0 0 :2 0 01 :1 0F3
0 .43 1 4S tirrin g o v e rn ig h t
2%
4 m l
8 0 0 :2 0 01 :1 0F4
0 .0 61 7 8S tirrin g o v e rn ig h t
5%
4 m l
8 0 0 :2 0 01 :2 0F5
D u a l D O X-C ro e n c a p su la tio n
P D IS iz e (n m )S o lv e n t re m o v a lP V AD C M (µ l):A c e to n e (µ l)D O X :C ro:P o ly m e rF o rm u la tio n
0 .22 5 6S tirrin g o v e rn ig h t
5%
4 m l
8 0 0 :2 0 01 :1 :2 0F6
Fig. 1. AFM image of PLGA-DOX-Cro NPs (F8) obtained in the tapping mode (A). Height profile of PLGA-DOX-Cro
NPs (B)
The cell viability of blank PLGA NPs was assayed
and exhibited no pronounce cytotoxicity to MCF-7
cells. Obtained results demonstrated that crocetin
could sensitize MCF-7 cells to doxorubicin-induced
cell death.As illustrated in Fig. 4 B and table 2, DOX
and crocetin in the PLGA-DOX-Cro NP exhibited
significantly higher cytotoxicity compared to other
formulations. However, PLGA-dox+cro and PLGA-
Dox could also decrease IC50 compared to
Doxorubicine (p valued”0.001). This may be due to
the different release rates of DOX and crocetin in
aqueous media or higher cellular uptake.Moreover,
because of high cellular uptake of NPs via
endocytosis, the co-encapsulation of DOX and
7. Nanomed. J., 3(1): 23-34, Winter 2016
29
crocetin in PLGA NPs was demonstrated more cytotoxic
acitivity against MCF-7 adenocarcinoma cell line in
comparison with free DOX and crocetin. On the other
hand, treatment with crocetin alone did not cause any
cytotoxicity, it was suggested that the enhancement
effect might result from the combination of individual
anti-tumor mechanism for each drug. DOX binds to
DNA by intercalation and promotes apoptosis in tumor
cells, and crocetin can act as chemosentizer or MDR
reverser. Codelivery of cytotoxic anti-cancer drugs with
natural product is considered as a solution for better
chemotherapeutic response in vitro and in vivo. As
the key point for successful combination therapy is to
design versatile codelivery systems.Previously, the
combination therapy of routine anti-cancer drugs with
different natural products which codeliver within PLGA
nanoparticle has been reported.Among this, curcumin
(active constituent of turmeric) is attracted much
attention and obtained results demonstrated its
synergistic effect along with doxorubicin, letrozol and
gemcitabine for cancerchemotherapy[49-51]. Until now
the combination therapy of DOX and crocetin was not
investigated in detail. In our study, crocetin increased
the cytotoxicity of DOX in MCF-7 cells in vitro. The
reason to prefer DOX and crocetin for co-encapsulation
was related to MDR reversibility of crocetin,
transmitting and maintenance of DOX inside the
nucleus. Then crocetin as a MDR reverser may help to
enhance antiproliferative activity of DOX in MCF-7
cells.
Fig. 2. In vitro release profile of DOX and Cro from PLGA
NPs in pH 7.4 at 37°C up to 48 hours
Fig. 3. 2-D and 3-D structures of crocetin
Appototic cell analysis by flow cytometry
A flow cytometeric analysis of propidium iodide
(PI)-stained cells was also performed to assess the
impact of crocetin on the generation of sub-diploid
cells with Dox. Co-treatment of MCF-7 cells with
crocetin in combination with doxorubicin increased the
sub-diploid population (fig. 5). Treatment with NPs
encapsulated both crocetin and doxorubicin
significantly increased (p<0.01) the sub-diploid
population for MCF-7 compared with free DOX+Cro.
Since, treatment with crocetin alone did not cause
increasing sub-diploid (p> 0.05) population for MCF-7
cells, suggesting crocetin is a qualified chemosentetizer
agent.
Obtained results are in agreement with the cell viability
data and verify the potency of the prepared PLGA NPs
in delivering the drugs to the cells, and codelivery of
DOX and crocetin through encapsulation in PLGA NPs
exhibited more anti-cancer activity.
Activation of cell death-associated caspases
In our study, increase in expression of caspase-3
as an executioner of cell apoptosis pathway, might play
a pivotal role in sensitizing MCF-7 cells to DOX-
induced apoptosis.Compared to the untreated cells,
the activity of caspase-3 in the MCF-7 cells treated
with nanoparticles containing Dox (DOX=1 µM) was
increased (Fig. 6). In comparison with free DOX+Cro,
PLGA NPs encapsulated DOX and Cro caused higher
expression of caspase-3 in treated cells.
8. Nanomed. J., 3(1): 23-34, Winter 2016
30
F. Alebooye Langroodi et al.
Table 2. IC50 of DOX in Solution or in formulations in the
presence or absence of Cro
*** p<0.001; ** p<0.01, *p<0.05
Fig. 5. % of sub G1 cells in MCF-7 cells after exposure to
free DOX, PLGA-DOX, PLGA-DOX-Croc, PLGA-
DOX+Cro, DOX+Cro (DOX concentrations were equivalent
to 0.5, 1, 2, 4 and 8µM and Crocetin in terms of the
different concentration existed in PLGA-DOX-Cro NPs)
*** p<0.001; ** p<0.01
Fig. 4. Cytotoxicity of different concentrations of Cro (A); free DOX, blank PLGA NPs, free DOX+Cro, PLGA-
DOX, PLGA-DOX+Cro and PLGA-DOX- Cro (B) in MCF-7 cell line for 48 hours. The Dox and Cro concentrations
in these formulations were 0.5-10 µM and 1-16 µM respectively
I C 5 0 ( µ M )F o r m u l a t i o n s
7 . 8 1 µ M* * *D O X
6 . 4 3 µ M* * *D O X + C r o
3 . 2 1 µ M* *P L G A- D O X
2 . 1 9 µ M *
P L G A- D O X N P s + f r e e
C r o
0 . 8 2 µ MP L G A- D O X- C r o
Fig. 6. Western blot analysis of caspase-3 following 48h
exposure of MCF-7 cells with DOX, DOX+Cro, PLGA-
DOX, PLGA-DOX+Cro and PLGA-DOX-Cro
Cellular uptake
To investigate the delivery of cargos via PLGANPs,
we evaluated the cellular uptake of drugs inMCF-7
using flow cytometry.While the uptake of drugs was
determined at 2 hrs, 24 hrs and 48 hrs, our studies show
that there was continued uptake of PLGA NPs for at
least up to 48 hrs in MCF-7 cells (Fig. 7). This suggests
the potential for even further improvement in
intracellular delivery of drugs with longer incubation
times.As illustrated in Fig. 6 PLGANPs in the presence
of crocetin enhances doxorubicin delivery to the cells
after 48 hrs. Fluorescent intensity of MCF-7 cells
incubated with PLGA-Dox-Cro was higer than PLGA-
DOX+ free Cro. Free DOX or Cro enters to the cells
through passive diffusion whereas NPs encapsulated
Cro/DOX enter into the cells by endocytosis that
results in higher uptake of drugs through NPs in
comparison to free drug. Presence of crocetin as a
carotenoid along with DOX in formulation prevents
9. Nanomed. J., 3(1): 23-34, Winter 2016
31
the efflux of Dox in MDR MCF-7 cells overexpressing
ABCB1 [52, 53]. Due to this reason the fluorescence
intensity of the cells treated with DOX along with
crocetin increased. In consistent with other reports,
our studies demonstrate that PLGA nanoparticle
encapsulation of hydrophilic drugs like doxorubicin
can significantly improve its therapeutic effect [54-57].
Additionally, codelivery of a potent anti-cancer natural
product such as crocetin along with doxorubicin could
result in further enhanced therapeutic efficacy.
Fig. 7. Flow cytometry detection of drugs uptake using
fluorescence intensity of the cells after 2h (A) , 24h (B) and
48h(C) incubation with free DOX, DOX+Cro, PLGA-DOX,
PLGA-Dox+Cro and PLGA-DOX-Cro NPs in MCF-7 cells
CONCLUSION
The effect of crocetin on antitumor activity of
doxorubicin encapsulated in PLGA nanpoparticles was
investigated against MCF-7 cells. Experiments on in
vitro drug release and cellular uptake of the PLGA-
DOX-Cro formulation indicated that DOX and Cro were
efficiently taken up by the cells and released
simultaneously. Furthermore, the codelivery of DOX/
Cro through PLGA NPs inhibits MCF-7 growth more
effectively than the delivery of either DOX or Cro at
the same concentrationsIt was proved that the
codelivery of DOX and Cro cause apoptosis induction
and also increase expression of caspase-3 protein.
We also suggested a possible mechanism enhancing
the tumor regression rate for the synergistic
therapeutic effect of DOX and Cro. In conclusion, the
PLGA NPs containing DOX and Cro have the potential
for future clinical application.
COMPETINGINTERESTS
The authors declare that they have no competing
interests.
ACKNOWLEDGEMENTS
The authors are grateful for the financial support
received from the Research Council of Mashhad
University of Medical Sciences.
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How to cite this article:
Alebooye Langroodi F, Hafezi Ghahestani Z, Alibolandi M, Ebrahimian M, Hashemi M. Evaluation of the effect of crocetin on antitumor
activity of doxorubicin encapsulated in PLGA nanoparticles. Nanomed. J., 2016; 3(1): 23-34.
DOI:10.7508/nmj.2016.01.003
URL:http://nmj.mums.ac.ir/article_6193_888.html
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