ITRM20150162A1 - ANTI-NEOPLASTIC ACTIVITY OF NANOVESCICULES ISOLATED BY CITRUS LIMON - Google Patents

Description

Title: Activities antineoplasty of nanovesicles isolated from Citrus Limon

Technical field of the invention

The present invention relates to the obtaining of vegetable products having activity? pharmacological and their therapeutic use. In particular, the invention refers to nano-sized vesicles obtained from the juice of plants of the Rutaceae family.

State of the Prior Art

Cancer represents, after the diseases of the cardiovascular system, the second cause of death in industrialized countries. There are more? of 100 different cancer isotypes including lung, colon, breast, pancreas, liver and leukemia. Surgery, chemotherapy and radiotherapy are the most important ones. common chemotherapeutic approaches adopted for the treatment of cancer patients. Data from the World Health Organization? they estimate that the average survival 5 years after diagnosis is about 60% despite the progress made in therapy and the considerable investments made in the pharmaceutical field. Conventional chemotherapy treatment often causes toxic effects on normal cells.

Therefore, there? an urgent need to identify new compounds and / or treatments more? effective in order to 1) inhibit the growth and progression of cancer and 2) have few side effects on normal cells.

Exosomes are small vesicles released by animal cells in the extracellular side. Exosomes are 30-100 nm in size and are present in many biological fluids. AND? These vesicles are known to carry the molecular products of the cell that produces them, including proteins, lipids, mRNA and microRNA (miRNA). Numerous data show that exosomes play an important role in cell-to-cell communication by influencing physiological and pathological processes. Previous studies suggest that nanovesicles released from plant cells are similar to exosomes (1). Zhang and colleagues reported that nanovesicles derived from edible plants such as grapes, grapefruit, ginger and carrots exhibit properties. anti-inflammatory in chronic inflammatory bowel diseases (2, 3). Zhang et al. they also reported in the US patent application US2014 / 0308212 the use of vegetable vesicles as carriers for medicaments, including anticancer drugs, encapsulated in them or as carriers that incorporate labeling molecules that can be used in diagnostic methods. However, the potential antineoplastic role of such plant-derived nanovesicles in influencing cancer progression is not? never been investigated until now.

Data in literature have shown that aqueous compounds and / or extracts from different varieties? plants, including Citrus limon L. (Rutaceae family), carry out an activity? antiproliferative and antitumor (4, 5) essentially linked to the presence of flavonoids. Therefore, an alternative approach to overcome the side effects of chemical compounds could be the use of natural agents, which provide an excellent contribution to modern therapies. Furthermore, ? It is known that some natural compounds are able to inhibit the development of cancer in animal models of carcinogenesis (6). ? it is also known that the TNF-related apoptosis-inducing ligand (TRAIL) protein induces apoptosis by binding to specific receptors (TRAIL-R). Such a family? emerged as a key mediator of cell survival, initiating cells towards the extrinsic pathway of apoptosis (7). ? important to emphasize that, unlike many chemotherapy drugs, TRAIL has the ability? to induce apoptosis in tumor cells but not in normal cells (8).

Purpose of the present invention? therefore that of providing new pharmacological tools that offer greater efficacy in inhibiting the development and progression of neoplasia and that at the same time have few side effects on the organism.

Summary of the Invention

The use of natural substances in the treatment of neoplasms currently represents a therapeutic alternative to the use of common chemotherapy drugs.

The present invention is based on the discovery that nanovesicles of vegetable origin obtained from the juice of plants of the Rutaceae family, specifically of the subfamily Aurantioideae, especially of the genus Citrus, exert a cytotoxic and / or cytostatic and / or sensitizing effect in the treatment of various neoplasms. Furthermore, the presence of said vesicles in the juice of plants of the genus Citrus is not? never previously been demonstrated.

Therefore the objects of the present invention are:

A process of obtaining nanovesicles from the juice of the fruits of the plant of the genus Citrus comprising the following steps:

a) centrifuge and filter the juice into one or more? consecutive cycles;

b) the juice obtained in step (a) is ultracentrifuged to obtain a supernatant liquid and a sedimentation pellet containing the nanovesicles and the vesicles are recovered.

The procedure can? also include the following additional steps:

c) the recovered pellet is subjected to ultracentrifugation in sucrose gradient; d) the fraction having density? between 1.12 and 1.19g / ml;

e) optionally the fraction is subjected to ultracentrifugation; And

f) optionally the fraction is washed with physiological solution.

A procedure in which ultracentrifugation in step (b) and / or (e)? performed at a value of g (i.e. acceleration of gravity) between 100.000xg and 150.000xg for a time between 120 and 60 min.

A procedure in which the? sucrose gradient ultracentrifugation at step (c)? performed at a value of g (acceleration of gravity?) between 100.000xg and 150.000xg for a time between 60 min and 120 min at a density gradient? between 30% and 45%. A process in which step (a) comprises at least one centrifugation at a g value between 15,000x g and 20,000x g for a time between 60 and 180 min and filtration with a pore filter not less than about 0.45 µm (micron).

A process in which step (a) comprises a first cycle of centrifugations from 3000xg to 10,000xg for a time between 30 and 90 min / centrifugation followed by a first filtration with a pore filter not less than 0.8 microns, and a second cycle of centrifugations from 15000xg to 17000xg for a time between 70 and 100 min / centrifugation followed or intercalated by filtrations with a pore filter not less than about 0.45 micron. They represent further objects of the invention:

Nanovesicles having density? between 1.12 and 1.19g / ml and dimensions between 50 and 70 nm obtained with the process of the invention for use in a therapeutic treatment as a single active ingredient or in combination with one or more? additional active ingredients, provided that one or more? additional active ingredients are not encapsulated in the nanovesicles themselves.

The above views nanovesicles for use in a preventive or curative therapeutic treatment of solid or hematological tumors, particularly for use in a treatment where the tumor? cancer of the colorectal, pancreas, lung, kidney, ovary, liver, thyroid, bladder, breast, esophagus, skin, acute leukemia, chronic myeloid leukemia or multiple myeloma; advantageously for use in a treatment where cancer? resistant to common chemotherapy.

They represent a further object of the invention:

The nanovesicles of the invention for use in a sensitization treatment of tumor cells to a subsequent or concomitant treatment with one or more? chemotherapeutic agents, advantageously for sensitizing tumor cells resistant to common chemotherapeutic agents.

A further object of the invention is a pharmaceutical composition containing the nanovesicles of the invention and a pharmaceutically acceptable excipient.

The invention described here is based on the identification in the juice of plants, preferably of the genus Citrus, for example of Citrus limon L., of a selected fraction of vesicles obtained following subsequent centrifugation and filtration, which has a high activity antitumor in vitro on tumor cell lines both sensitive and resistant to the common chemotherapeutic agents used today.

To date, the anticancer therapies in use for the treatment of different solid and haematological neoplasms involve the use of substances (chemotherapy) that induce cytotoxicity. not only in cancer cells but also in normal cells. Furthermore, although chemotherapy treatment has had high success rates for the treatment of various neoplasms, the acquisition of drug resistance is today one of the crucial aspects in the treatment of cancer. For example, the acquisition of drug resistance in patients with chronic myeloid leukemia, observed with an annual frequency of 4%, has in recent years posed the need? to develop new therapeutic strategies.

The main advantage that the invention object of the present application offers lies in the use of plant-based nanovesicles, the content of which is non-toxic for normal cells, inducing instead the arrest of the proliferative process of tumor cells, starting them towards death.

The nanovesicles isolated from vegetable juice can be used as single therapeutic agents, or in combination with other chemotherapeutic agents or to sensitize tumor cells to treatment with common chemotherapeutic agents. However, ? It is important to note that the nanovesicles of the invention independently carry out an activity? antitumor and even when they are administered in combination therapy with other drugs, their therapeutic function does not? that of the carrier that carries or encapsulates said additional medicaments, but that of the autonomous active principle, which exercises a complementary, additional or synergistic therapeutic function.

Description of the Figures

Figure 1: the figure illustrates the characterization of nanovesicles by electron microscope (figure 1a), analysis by DLS (dynamic light scattering, figure 1b) and by proteomic approach (figure 1c) and the internalization of vesicles in human tumor cells A549 and LAMA84 ( figure 1d).

Figure 2: the figure illustrates the activity? antiproliferative of nanovesicles on colon cancer (SW480), lung (A549, CRL2868, CRL5908 resistant to erlotinib and gefitinib) and chronic myeloid leukemia sensitive and resistant to imatinib (LAMA84S, LAMA84R).

Figure 3: The figure illustrates the expression of several molecules involved in the apoptotic pathway. As shown in Figure 3a, the A549, SW480 and LAMA84 cells treated with the nanovesiocles show an increase in the expression of the pro-apoptotic genes, Bad and Bax, and a reduction in the mRNA levels of the anti-apoptotic genes Survivin and Bcl-XL. The increase in BAX protein expression and the decrease in BCL-xL were also confirmed by western blot analysis (Figure 3b).

Figure 4: The figure illustrates the increase in gene expression of TRAIL (Figure 4a, upper panel) and DR5 (Figure 4a, lower panel) induced by the nanovesicles of the invention. Increased release of the TRAIL protein? was also confirmed by the ELISA test, as shown in Figure 4b.

Figure 5: The figure illustrates the capacity? of nanovesicles to inhibit tumor growth in an in vivo model of chronic myeloid leukemia.

Figure 5a shows that tumor growth? delayed in mice treated with nanovesicles, both locally and intraperitoneally (left panel), leading to the formation of more tumors. small compared to control mice (right panel).

Figure 5b illustrates the increase in the proapoptotic genes Bad and Bax and the decrease in the antiapoptotic genes, Survivin and Bcl-XL and the increase in TRAIL and DR5 in mice treated with nanovesicles. Figure 5c shows the increase in the number of TRAIL positive cells (indicated by arrows) in tumors of locally or intraperitoneally treated mice compared to control mice.

Figure 6: The figure illustrates the biodistribution of the labeled nanovesicles in the tumor site (figure 6a - arrows indicate the labeled nanovesicles localizing around the tumor). Analysis of the tumors shows that the nanovesicles are internalized in the tumor mass and remain in the site, while no accumulation of fluorescence is observed in the tumor taken from the mouse treated with the free probe (Figure 6b).

DETAILED DESCRIPTION OF THE INVENTION

The vegetable vesicles of the invention are obtained from the fruit juice of plants of the Rutaceae family, specifically of the subfamily Aurantioideae, and particularly of the genus Citrus, for example, Citrus limon L. (lemon), Citrus medica (cedar), Citrus reticulata ( mandarin), Citrus paradisi (grapefruit), Citrus sinensis (sweet orange).

The procedure

The procedure for obtaining the nanovesicles of the invention provides for successive steps of selection and isolation by means of centrifugation and filtration techniques, until a specific selected fraction is obtained. Said isolated fraction? characterized by a density? between 1.12 and 1.19 g / ml and a proteomic profile that for 56.7% of the identifications, consists of proteins already? described in mammalian exosomes. The process includes a preparatory phase (a) of the juice in which the juice is subjected in one or more? consecutive cycles of centrifugation and filtration. This stage? aimed at removing from the juice, by sedimentation and / or filtration every solid component and every element of the high density juice.

The procedure then includes the following step (b) and optionally the steps (c), and (d) performed on the juice treated in the preparatory phase:

b) the juice obtained in step (a) is ultracentrifuged to obtain a supernatant liquid and a sedimentation pellet containing the nanovesicles.

The step of ultracentrifugation (b)? performed at a value of g (acceleration of gravity?) between 100.000xg and 150.000xg for example at 110.000xg, 120.000xg, 130.000xg, 140.000xg and for a time inversely proportional to the g value, that is? between 120 and 60 min. In a specific embodiment yes? chosen for example 120.000xg for 90 minutes or 130.000xg for 80 min. The ultracentrifugation steps are performed at a refrigerated temperature, preferably below 10 ° C for example at 4 ° C. The supernatant liquid? discarded and the pellet settled? recovered. The pellet sedimented so? obtained containing the nanovesicles pu? be used for the therapeutic purposes described here. For illustrative and non-limiting purposes, ultracentrifugation can? be carried out using the Optima ultracentrifuge? L-80 XP - Beckman Coulter with Type 70 T1 rotor.

c) In order to obtain the nanovesicles in a purified form and with greater activity? biological, you can? subject the recovered pellet to ultracentrifugation in sucrose gradient.

Such a sucrose gradient ultracentrifugation? performed at a value of g included (between 100.000x g and 150.000x g for example at 110.000xg, 120.000xg, 130.000xg, 140.000xg and for a time inversely proportional to the g value, i.e. between 120 and 60 min. on a sucrose gradient with density between 30% and 45%.

In particular, the 30% to 45% sucrose solutions are placed at the bottom of an ultracentrifuge tube and the pellet of step b) comprising the vesicles? layered on the sucrose solution. For illustrative and non-limiting purposes, ultracentrifugation can? be carried out using the Optima ultracentrifuge? L-80 XP - Beckman Coulter with SW28 rotor .. After ultracentrifugation a sucrose buffer is formed which includes the nanovesicles with high density. between 1.12 and 1.19g / ml.

d) Finally, the fraction having preferably density? comprised between 1.12 and 1.19g / ml and comprising the nanovesicles of the invention.

Subsequently, in order to eliminate the sucrose residues present in the preparation, the isolated fraction pu? undergo an ultracentrifugation step (e) under the same conditions described for step (b). Preferably, ultracentrifugation? carried out at 100.000xg for about 90 minutes at low temperature: e.g. 4? C.

Finally, the pellet sedimented in step (e) can? be washed one or more? times with physiological solution.

What about the preparatory or preliminary phase (a)? evident that this can? include any combination of centrifugation and filtration, of? g? values, of centrifugation times and porosity? filtering, to the extent that? this combination allows to remove from the juice every solid component and every element of the high density juice.

In particular, this phase necessarily includes one or more? centrifugation passages at a g value between 15,000 and 20,000 followed or alternated with one or more? filtrations. Each centrifugation will be able? be performed for example at 15.500xg, 16.000xg, 16.500xg, 17.000xg, 17.500xg, 18.000xg, 19.000xg, for a time inversely proportional to the value? g?, between 240 and 60 min per centrifugation. Are the centrifugation steps alternated with filtration steps with porosity filters? between approximately 0.80? m (microns) and approximately 0.45? m (microns). In one embodiment of the invention step (a) will comprise? a first centrifugation for example at 16500xg for a time of 60-90 minutes, followed by a filtration with pores not lower than about 0.45 microns followed by a second centrifugation at the same value of? g? for a longer time? long, for example 180-240 minutes.

The preparatory phase (a) can? still understand one or more? preliminary centrifugation cycles at a low value of? g? to remove the more solid materials from the juice? coarse or the elements of the juice to density? greater. This phase can? also include one or more? filtration steps. Are centrifugations performed at value? G? between 2500xg and 11.000xg. In a specific embodiment, more than one can be envisaged. consecutive steps in value? g? increasing, for a period of between 20 and 90 minutes by centrifugation. For example, you can? perform a first centrifugation at 2500xg, 3000xg or 3500xg for 20-30 minutes followed by a second centrifugation at 9500g, 10.000g or 10.500g for 55-70 minutes. The centrifugation cycle can? be followed by filtration on a porosity filter? not less than 0.80 microns. ? clear that after each centrifugation step in step (a), the supernatant will be? collected and subjected to the following steps, while the sedimented materials or fractions will be discarded.

An exemplary embodiment of the process of the invention as a whole? ? described in the experimental part in the chapter entitled? Preparation of the nanovesicles ?.

The quantification of nanovesicles? was determined by Bradford assay.

The vesicles

The nanovesicles of the invention, obtained through the procedure described above, are characterized by a density? in sucrose gradient, between 1.12 and 1.19 g / ml and a specific proteomic profile. The plant nanovesicles of the invention are comparable to the typical exosomes of mammalian cells. They too have a diameter ranging from about 30 to about 100 nm, plus? frequently between 40 nm and 70 nm, for example 50 nm or 60 nm. (Dimensions are determined by dynamic light scattering (DLS) analysis as described in Viktoriya Sokolova et al. (9). Nanovesicles also exhibit morphology similar to that of mammalian exosomes: that is, small extracellular vesicles consisting of a lipid bilayer. , from proteins and containing nucleic acids inside them. From the proteomic characterization it emerges that about 56.7% of the identifications, consists of proteins already described in mammalian exosomes and belonging to the following protein functional groups: proteasome, heat shock proteins, cytoskeleton , ubiquitin system, transporters, related amino acid enzymes, RNA transfer biogenesis, ribosomal proteins, translation factors, metabolic enzymes, phosphatases and associated proteins, kinases, GTP binding proteins, proteins involved in vesicle biogenesis and membrane trafficking They are also characterized by a specific protein profile which has been analyzed using two different proteomic approaches: gel-free and gel-based. The analyzes were carried out using the Triple-TOF 5600 Plus and showed that the proteins contained in the vesicles of the invention are only homologous to those contained in the mammalian exosomes. In particular, the higher proteins? frequent in the nanovesicles described herein are included in the group comprising the subunit? regulatory 5 non-ATPase of the 26S proteasome (K06692); ARF1 protein (K07937); annexin A7 / 11 (K17095), aquaporin PIP (K09872), subunit? alpha / atp1 of ATP synthase (E5DK62); subunit? beta of the ATP synthase transporter H <+> type F (K02133); ABCF1 protein (K06184); citrate synthase (K01647); 4G translation start factor (K03260); elongation factor 1-beta (K03232); 1-gamma elongation factor (K032339; elongation factor 2 (K03234); TSG101 subunit of ESCRT-I complex (K12183); VPS28 subunit of ESCRT-I complex (K12184); VPS37 subunit of ESCRT-I complex ( K12185); translation initiation factor 2C (K11593); exportin-1 (K14290); exportin-2 (K18423); protein HSP70 (K03283); protein HSP90 (F1CGQ9); protein HSP20 (K13993); ribosomal protein L10e (K02866 ); ribosomal protein L13A (K02872); lysophospholipase II (K06130), plant SNARE (K08494); phospholipase C (K01114); protein SEC31 (K14005); protein Rab-11A (K07904);; protein Rab-7a (K07897); serpin B (K13963); ribosomal protein S12e (K02951); ribosomal protein S5e (K02989); ribosomal protein S7e (K02993); ribosomal protein S9e (K02997); succinate dehydrogenase subunit ((K00234); translation initiation factor 4A (K03257); transporter-1 (K18752); ubiquitin-C (K08770); ubiquitin activator (E1) (K03178); protein VPS35 (K18468); protein V PS4 (K12196); interaction protein with t-SNAREs 1 (K08493); vesicle membrane associated protein 7 (K08515).

Activities therapeutic

The data reported below show that the nanovesicles isolated from the fruit juice of plants of the genus Citrus, such as for example the Citrus Limon L. (family Rutaceae) have activity? antineoplasty in vivo on animal model and in vitro, on a panel of different cell lines of solid and haematological tumors, such as colorectal cancer (adenocarcinoma), pancreatic, lung, kidney, ovarian, liver, thyroid cancer , bladder, breast, esophagus, skin; leukemia; chronic myeloid leukemia and multiple myeloma.

Surprisingly, the vesicles of the invention proved to be active not only on sensitive tumor cell lines, but also on lines resistant to common chemotherapeutic agents, such as erlotinib or gefitinib, as well as on lines resistant to common chemotherapeutic agents, such as erlotinib or gefitinib. in an in vivo model of imatinib-sensitive or resistant chronic myeloid leukemia (CML).

Therefore, the nanovesicles of the invention find application in the preventive and curative treatment of a wide spectrum of neoplasms both as single therapeutic agents capable of autonomously developing a therapeutic effect, and in combination with other chemotherapeutic agents in order to obtain an additional, complementary or synergistic, both in a pre-treatment of sensitization of tumor cells to a subsequent treatment with a chemotherapy, especially tumor cells resistant to the chemotherapy itself.

Without wishing to link and limit the invention to specific theories, the results achieved show that the nanovesicles of the invention induce the arrest of tumor cell proliferation by activating the extrinsic pathway of apoptosis mediated by the TRAIL protein, both in vitro and in I live. TRAIL induces apoptosis in target cells expressing the DR5 death receptor, without affecting viability. of normal cells. Activation of the TRAIL-mediated pathway leads to an increase in the expression of pro-apoptotic genes (Bad, Bax) and a decrease in anti-apoptotic genes (Bcl-xl, survivin). TRAIL induces apoptosis preferentially in tumor cells, but not in normal cells. AND? It was initially proposed that the induction of TRAIL-mediated apoptosis, preferentially in tumor cells, is due to the fact that the TRAIL-R1 and TRAIL-R2 death receptors are mainly expressed in these cells, hence the regulation of TRAIL-induced apoptosis it could, at least in part, be determined by the expression of said receptors.

[Taken together, these results highlight that the use of nanoparticles from Citrus limon represents an alternative approach to cancer treatment. The natural origin of their content is non-toxic for normal cells, instead inducing the arrest of the proliferative process of cancer cells, starting them towards death.]

EXPERIMENTAL PART

Preparation of the nanovesicles

For example, the nanovesicles have been isolated from the juice of Citrus limon L. The juice? was first centrifuged at 3000g for 30 minutes, then at 10000g for 1 hour. The supernatant? it was then subjected to filtration with filters with 0.8 µm pores and centrifuged at 16500xg for 1 hour. The supernatant? was again subjected to filtration with 0.45µm pore filters and centrifuged at 16500xg for 3 hours. The supernant? it was then ultracentrifuged for 90 minutes at 120000xg. The pellet cos? sedimented and containing the nanovesicles? was collected and subjected to ultracentrifugation at 100,000 g (centrifugal force) in a sucrose gradient at 30-45% sucrose, for 90 minutes. Subsequently ? has been recovered the fraction of density? between 1.12 and 1.19g / ml, subjected to ultracentrifugation and then washed more? times in PBS. The quantification of nanovesicles? was determined by the Bradford assay described below.

Essentially identical vesicles were obtained by removing centrifugation at 3000xg, and centrifuging at 10,000xg for 90 minutes; or by performing the first centrifugation at 2500xg for 40 minutes or at 3500xg for 20 min; or by performing the second centrifugation at 9500xg for 80 minutes or at 10500xg for 50 minutes; or the third centrifugation at 15500xg or 17500xg for 70 and 50 minutes respectively; or the fourth centrifugation equally at 15500xg or 17500xg for 200 minutes and 150 minutes respectively.

Essentially identical vesicles were obtained by conducting l? ultracentrifugation at 110,000xg or 130,000xg for 110 and 80 minutes respectively.

Quantification of nanovesicles by Bradford assay

In particular, this assay allows to know the concentration of the protein extract thanks to a colorimetric reaction due to the interaction of the Coomassie solution with specific amino acids (histidine, arginine and aromatic amino acids); this bond produces a color change, from brown to blue, in proportion to the quantity? of proteins present in the extract.

Such an essay? performed by diluting the selected fraction of vesicles in PBS, adding the Coomassie solution and evaluating the absorbance at the wavelength of 595 nm. Preferably,? made a 1: 5 dilution of the selected fraction in PBS. About 50 µl of this dilution are then added to 1.5 ml of the Coomassie protein assay kit solution (Pierce, Rockford, IL, USA) and the absorbance evaluated by the Biophotometer at the length d? wave of 595 nm. The values obtained are zeroed against the blank (1.5 ml of the Coomassie protein assay kit solution alone and 50 µl of PBS). The final concentration in? G /? L? obtained by dividing the mean absorbency value of each sample by the slope value of a BSA (bovine serum albumin) curve, previously constructed from serial dilutions of a known concentration of the protein; the value obtained? multiplied by the dilution factor.

The vesicles were characterized by electron microscope (Figure 1a): the nanovesicles resuspended in PBS were plated on a carbon-coated grids and stained with 2% phosphotungstic acid. The preparation obtained? visualized using a transmission electron microscope JEOL JEM-1400 Plus transmission electron microscope, a110kV.

The vesicle sizes (50-70 nm) were analyzed by DLS (dynamic light scattering, Figure 1b) and by proteomic approach (Figure 1c). As shown in Figure 1, the isolated nanovesicles represent a homogeneous population in size; furthermore, the analysis of the protein composition of the vesicles reveals the presence of proteins homologous to the proteins characteristic of mammalian exosomes. Equivalent vesicles were obtained through the same procedure performed on Citrus medica (cedar), Citrus paradisi (grapefruit).

Internalization test in tumor cells

Cell lines:

Chronic myeloid leukemia cells LAMA84 and LAMA84R (resistant to imatinib), colon adenocarcinoma SW480 and lung adenocarcinoma A549, CRL2868, CRL5908 (resistant to erlotinib and gefitinib), were cultured in RPMI 10% FBS. HS5 stromal cells were cultured in DMEM 10% FBS, HUVEC endothelial cells in Endothelial Growth Medium (EGM). PBMC cells were isolated by Ficoll Paque separation technique.

Evaluation of the internalization of Citrus nanovesicles in A549 and LAMA84 cells

In order to determine whether lemon vesicles are internalized by human cancer cells, the nanovesicles were labeled with the lipophilic dye PKH26 (red) for 10 minutes at room temperature. A549 and LAMA84 cells were seeded in collagenated slides and treated with 20 µg / ml of labeled nanovesicles for 3 and 6h. The cells were then labeled with Actin green (green-actin) and Hoechst (blunuclei) and analyzed under a confocal microscope. As shown in Figure 1d, A549 cells (human lung adenocarcinoma epithelial cells) and LAMA84 chronic myeloid leukemia cells, treated for 3 and 6 hours with lemon vesicles internalize the lemon vesicles (arrows indicate cells that internalized the marked vesicles) with time dependent efficacy.

Activity test antiproliferative

Sage of vitality? MTT cell phone

The vitality? cell phone ? was evaluated by MTT assay. Cells were seeded in 96-well plates and treated for 24,48 and 72h with 5-20? g / ml of nanovesicles. L? MTT? a vital dye which, upon entering cells, is reduced by mitochondrial dehydrogenase. For each well containing 100 µl of cell suspension, 10 µl of MTT was added and the plates were kept for 4 hours in an incubator at 37 ° C. At the end of the incubation, the cells were lysed by adding 150 µl of isopraponol-HCl 0.05M per well with consequent leakage of the dye from the cells themselves. The mitochondrial enzyme? active only in living cells, and its function consists in cutting the tetrazolium ring of MTT (yellow substance) with the formation, consequently, of formazan (a blue salt). Therefore, the intensity? of coloring? proportional to the number of live cells and is evaluated by means of an ELISA plate reader at a wavelength of 540 nm; the values of density? optics (O.D.) were zeroed against blank (100 µl of cell culture medium, 10 µl of MTT and 150 µl of isopropanol-HCl). As shown in Figure 2, the treatment of colon (SW480), lung (A549, CRL2868, CRL5908 resistant to erlotinib and gefitinib) and chronic myeloid leukemia cells sensitive and resistant to imatinib (LAMA84S, LAMA84R) with lemon nanovesicles , leads to a dose and time-dependent decrease in vitality? cell phone. The same effect is not observed in non-tumor cells (endothelial cells, stromal cells, peripheral blood mononuclear cells). Activity test apoptotic

RNA extraction and Real time PCR

LAMA84, A549 and SW480 were plated in 12-well plates and treated with 5 and 20? G / ml of nanovesicles for 24 and 48 hours. Tumor biopsies were stored in RNAlater (Applied Biosystems, Foster City, California, USA). RNA? was isolated using a commercial RNA spin Mini Isolation Kit (GE Healthcare). The total RNA? it was then back-transcribed in cDNA (High Capacity cDNA Reverse Transcription Kit, Applied Biosystem). The cDNA cos? obtained ? was subjected to quantitative Real time PCR using 48-well plates Step-One Real-Time PCR system (Applied Biosystem) using specific primers for the following genes:

The changes in the gene of interest were normalized with respect to the GAPDH content using the comparative method of ?? Ct to calculate the expression changes which were then expressed as fold of induction.

To evaluate the mechanism by which nanovesicles are able to influence tumor growth, we tested the expression of several molecules involved in the apoptotic pathway. As shown in Figure 3, A549, SW480 and LAMA84 cells treated for 24 or 48 hours with the nanovesciocles show an increase in the expression of the proapoptotic genes, Bad and Bax, and a reduction in the mRNA levels of the antiapoptotic genes Survivin and Bcl. -XL, especially after 48 hours of treatment.

Western Blot

A549, SW480, LAMA84 cells were treated for 48 h with 20? G / ml of nanovesicles. The protein lysate obtained? was analyzed by SDS-PAGE and Western Blotting. The antibodies used in the experiments are: anti-BAX, BCL-xL and? Actin (Santa Cruz Biotechnology, CA, USA).

The increase in expression of the BAX protein (A5494.5 fold, SW4801.65 fold, 3.6 fold in LAMA84) and the decrease in BCL-xL (decrease 0.7 fold in A549, 0.88-fold in SW480, 0.82-fold in LAMA84) were also confirmed by western blot analysis (Figure 3b).

Activation of TRAIL gene expression

The results reported here also show that treatment with nanovesicles induces an increase in the gene expression of TRAIL (Figure 4a, upper panel) and DR5 (Figure 4a, lower panel). Increased release of the TRAIL protein? was also confirmed by the ELISA test, as shown in Figure 4b. The protein concentration of TRAIL? was determined by ELISA kit (Uscn life Science Inc., Houston, TX, USA).

Overall, these data suggest that nanovesicles induce cell death in tumor cells by activating the TRAIL / DR5-dependent apoptotic pathway.

Activities in vivo antitumor model of chronic myeloid leukemia

In vivo xenograft model of CML

NOD / SCID mice mice (Charles River Laboratories International, Inc, MA, USA) were inoculated in the right flank with LAMA84 chronic myeloid leukemia cells (2 x 10 <7> cells in 0.2 ml of PBS). Seven days after inoculation, the mice were treated with lemon nanovesicles (50? G / mouse, 3 times a week for 2 weeks) both intraperitoneally (IP) and intramassly (IT). Tumors were measured 3 times per week. At the end of the treatment, the mice were sacrificed and the tumors collected for gene expression and immunofluorescence analyzes.

Immunofluorescence

Tumors were fixed in 10% formalin and embedded in paraffin. Sections of 5- µm were used for immunofluorescence analyzes. Sections were incubated with anti-TRAIL antibody (Abcam) and analyzed under a confocal microscope.

In vivo assay

The capacity of nanovesicles to inhibit tumor growth? was also tested in an in vivo model of chronic myeloid leukemia. LAMA84 cells were inoculated subcutaneously into NOD / SCID mice; one week after inoculation, mice were treated locally (intra tumor, IT) or intraperitoneally (IP) three times a week with vehicle (PBS) or lemon nanovesicles. At the end of the treatment, the mice were sacrificed and the tumors removed. Figure 5a shows that tumor growth? delayed in mice treated with nanovesicles, both locally and intraperitoneally (left panel), leading to the formation of more tumors. small compared to control mice (right panel). Real-time PCR analysis in isolated tumors in vivo confirm the data obtained in vitro; as shown in Figure 5b, an increase in the proapoptotic genes Bad and Bax and a decrease in the antiapoptotic genes, Survivin and Bcl-XL, are observed in mice treated with nanovesicles. We also observe the increase in TRAIL and DR5. The results were confirmed by immunofluorescence analysis for TRAIL; yes ? observed an increase in the number of TRAIL positive cells (indicated by arrows) in tumors of locally or intraperitoneally treated citrus mice compared to control mice (Figure 5c). These data suggest that nanovesicles are able to reduce tumor growth by activating the TRAIL-mediated apoptotic pathway.

Study of the capacity? targeting of the tumor mass

In vivo biodistribution of nanovesicles

The nanovesicles were labeled with DiR lipophilic dye for 30 minutes at room temperature. The labeled nanovesicles were injected intraperitoneally into mice with CML xenographs. The mice were anesthetized and the fluorescence acquired? was performed using IVIS (Living Image Software; PerkinElmer LifeSciences) 15 minutes, 1 hour or 24 hours after inoculation. After 24 hours the mice were sacrificed, the organs and tumors removed and the fluorescence measured.

In vivo assay

In order to evaluate whether lemon nanovesicles reduce tumor growth in vivo by reaching the tumor site, the nanovesicles were labeled with the lipophilic dye DiR (1,10-dioctadecyl-3,3,30,30-tetramethylindotricarbocyanine). The biodistribution of nanovesicles? was evaluated 15 minutes, 1 hour and 24 hours after intraperitoneal inoculation of the labeled vesicles. As shown in Figure 6a, the vesicles quickly reach the tumor site (already after 15 minutes), remaining associated with the tumor for up to 24 hours (the arrows indicate the distribution of the labeled nanovesicles in the tumor site). The analysis of the organs taken 24 hours after the inoculation of the nanovesicles shows that these, so? like the free probe, they accumulate in the liver, spleen and kidneys. Furthermore, the tumor analysis shows that the nanovesicles are internalized in the tumor mass and remain in the site, while no accumulation of fluorescence is observed in the tumor taken from the mouse treated with the free probe (Figure 6b).

Statistical analysis

All in vitro experiments were repeated at least three times in order to obtain a reproducible result. Are the data expressed as average values? standard deviation of three independent experiments. Statistical analysis? was performed by t-test. The differences were considered significant when p? 0.05.

Claims (15)

CLAIMS 1. Process of obtaining nanovesicles from the juice of fruits of the plant of the genus Citrus comprising the following steps: a) centrifuge and filter the juice into one or more? consecutive cycles; b) the juice obtained in step (a) is ultracentrifuged to obtain a supernatant liquid and a sedimentation pellet containing the nanovesicles and the vesicles are recovered. 2. Process according to claim 1 comprising the following further steps: c) the recovered pellet is subjected to ultracentrifugation in sucrose gradient; d) the fraction having density? between 1.12 and 1.19 g / ml; e) optionally the fraction is subjected to ultracentrifugation; f) optionally the pellet is washed with physiological solution. 3. Process according to claim 1 or 2 wherein the ultrafiltration at step (b) and / or (e)? performed at a value between 100.000xg and 150.000xg for a time between 120 and 60 min. 4. Process according to any one of claims 2 or 3 wherein the sucrose gradient ultrafiltration at step (c)? performed at a value between 100.000xg and 150.000xg for a time between 120 min and 60 min at a% sucrose gradient between 45% and 30%. 5. Process according to any one of the preceding claims wherein step (a) comprises at least one centrifugation at a value between 15,000x g and 20,000x g for a time between 60 and 180 min and a filtration with a pore filter not less than about 0.45? M (micron). 6. Process according to claim 5 wherein step (a) comprises a first cycle of centrifugations from 3000xg to 10,000xg for a time between 30 and 90 min / centrifugation followed by a first filtration with a pore filter not less than 0.8 micron, and a second centrifugation cycle from 15000xg to 17000xg for a time between 70 and 100 min / centrifugation followed or intercalated by filtration with a pore filter not less than about 0.45 micron. 7. Process according to any one of the preceding claims comprising the following cycles: a) the juice is centrifuged at 3000xg for about 30 minutes, then at 10000xg for about 60 minutes and the supernatant is subjected to filtration with filters with 0.8 µm pores; b) the filtrate of the previous step is centrifuged at a value of 16500xg for about 60 minutes, the supernatant is subjected to filtration with filters with pores of 0.45 µm and the filtrate is centrifuged again at 16500xg for about 180 minutes; c) the supernatant of the previous step is ultracentrifuged at 120000xg for about 90 minutes to obtain a supernatant and a sedimentation pellet containing the nanovesicles; d) optionally the pellet is recovered and ultracentrifuged in a sucrose gradient between 45% and 30% of sucrose, ultracentrifuging at a value of about 100,000x g for 90 minutes at 4 ° C, e) the fraction having density? between 1.12 and 1.19 g / ml, f) the fraction in step (e) is ultracentrifuged under the same conditions as in (c), g) optionally washed with physiological solution. 8. Process according to any one of the preceding claims wherein the juice? obtained from Citrus limon L. 9. Nanovesicles having density? between 1.12 and 1.19g / ml and dimensions between 50 and 70 nm obtained by the process according to any one of claims 1 to 8 for use in a therapeutic treatment as a single active ingredient or in combination with one or more? additional active ingredients, provided that one or more? additional active ingredients are not encapsulated in the nanovesicles themselves. 10. Nanovesicles for use in a treatment according to claim 9 wherein the treatment? preventive or curative treatment of solid or haematological tumors. 11. Nanovesicles for use in a treatment according to claim 10 wherein the tumor? cancer of the colorectal, pancreas, lung, kidney, ovary, liver, thyroid, bladder, breast, esophagus, skin, acute leukemia, chronic myeloid leukemia and multiple myeloma. 12. Nanovesicles for use in a treatment according to claims 10 or 11 wherein the cancer? resistant to common chemotherapy drugs such as erlotinib, gefitinib or imatinib. 13. Nanovesicles for use in a treatment according to claim 9 wherein the treatment with the nanovesicles? a tumor cell sensitization treatment to a subsequent or concomitant treatment with one or more? chemotherapy. 14. Nanovesicles for use in a treatment according to claim 13 wherein the treatment with the nanovesicles? a sensitization treatment of cancer cells resistant to common chemotherapy. Pharmaceutical composition containing the nanovesicles according to any one of claims 9 to 14 and a pharmaceutically acceptable excipient.