Drug delivery system

Abstract

To provide a delivery system for molecules, preferably drugs.SOLUTION: The system comprises fat tissue or derivatives thereof. Moreover, the present invention refers to a fat-based delivery system, preferably loaded with molecules having antitumor activities, for use in the treatment of cancers.SELECTED DRAWING: Figure 3

Description

[Technical Field] 【0001】 explanation The present invention relates to a delivery system for molecules, preferably drugs, said system comprising adipose tissue or a derivative thereof. Moreover, the present invention relates to lipid-based delivery systems, preferably loaded with molecules having anti-tumor activity, for use in the treatment of cancer. [Background technology] 【0002】 background Drugs have long been used to improve health and extend lifespan. It is well known that the delivery approach has a significant impact on the efficacy of a drug. Indeed, some drugs are characterized by an optimal concentration range where maximum benefit is derived, and concentrations outside this range may be toxic or may not produce any therapeutic effect. 【0003】 Meanwhile, the very slow progress in disease treatment has reinforced the need for a multidisciplinary approach to the delivery of therapeutic agents to tissue targets. This need has driven dramatic changes in drug delivery practices over the past few decades, and even greater changes are expected in the near future. 【0004】 In particular, different concepts and delivery methods have recently been developed based on the use of cells as therapeutic carriers. Cell-based vehicles are particularly attractive for the delivery of biological therapeutics that are difficult to synthesize, have short half-lives, have limited tissue penetration, or are rapidly inactivated by direct in vivo introduction. The use of physiological carriers to deliver therapeutic agents systemically to improve their efficacy while minimizing adverse side effects is an attractive concept that could be applied in many clinical settings. 【0005】 The present invention proposes a delivery system made of adipose tissue or its derivatives as a solution to the previously reported needs, specifically the adipose tissue being lipoaspirate tissue, preferably microfragmented adipose tissue and / or microfragmented lipoaspirate tissue. 【0006】 In fact, the authors of the present invention have surprisingly found that: 1) Isolated fat tissue, preferably micro-fragmented fat tissue, delivery system (scaffold) is rapidly loaded with molecules / drugs (loading / priming phase takes minutes / hours); 2) the loaded molecules / drugs are released in therapeutically effective amounts and in a sustained manner; 3) the added molecules / drugs maintain their biological activity after release; 【0007】 4) decellularization (destruction of cellular components) of adipose tissue, preferably microfragmented adipose tissue, does not affect the release of molecules / drugs and, advantageously, means that the scaffolds disclosed in this context, to which molecules / drugs are eventually added, can also be kept frozen and used in subsequent steps; 【0008】 5) A wide range of molecules / drugs can be added to such a type of delivery system; 6) The delivery system and its degradation products of the present invention are biologically compatible and are not associated with toxic effects; and 7) The delivery system of the present invention is a natural part of the body and therefore is not immunogenic except for being biodegradable. 【0009】 Specifically, the previously reported effects were experimentally demonstrated by using both the aspirated fat tissue itself and microfragmented aspirated fat tissue, i.e., the adipose tissue obtained after the controlled microfragmentation process of the aspirated fat tissue disclosed below. 【0010】 However, a microfragmented fat delivery system is more advantageous than lipoaspirate (adipose tissue) itself because it is easier to handle and it allows for better standardization and reliability of treatment results. In this regard, it is well known how important standardization and reliability are for any treatment application. Summary of the Invention 【0011】 A first aspect of the present invention relates to a tissue-based delivery system for molecules, preferably drugs, wherein said tissue is isolated adipose tissue or a derivative thereof, or preferably lipoaspirate tissue. Preferably, the tissue is microfragmented fat or microfragmented lipoaspirate tissue, preferably isolated from any animal, more preferably it is isolated from a human, said human being being alive or dead, and / or preferably comprises clusters of adipose tissue having a size ranging from 10-5000 μm, more preferably 100-3000 μm, even more preferably 200-2500 μm, more preferably 300-1500 μm, more preferably 400-900 μm. 【0012】 Preferably, the microfragmented lipid / adipose tissue and / or clusters of microfragmented lipid / adipose tissue comprise cells selected from: mesenchymal stem cells (MSCs), adipose-derived stem cells (ASCs), adipose stem cells, pericytes, adipocytes, endothelial cells, and any combination thereof. Preferably, the molecule / drug is selected from: an anti-inflammatory molecule, an antibiotic, an anti-cancer molecule, and a 5α-reductase inhibitor. 【0013】 Anti-cancer molecules (chemotherapeutic agents) are preferably selected from: natural products, preferably vinca alkaloids, more preferably: vinblastine, vincristine, and vinorelbine, taxanes, preferably paclitaxel or docetaxel, vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxin (teniposide), actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine Selected from oxaliplatin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16), adriamycin, amsacrine, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, pemetrexed, 5-FU, rafenib, methotrexate, cyclophosphamide, bortezomib, tomozolomide, sorafenib, and any combination thereof. 【0014】 More preferably, the anti-cancer molecule is selected from: paclitaxel (PTX - Taxol or Onxar) or a derivative thereof, preferably Abraxane, docetaxel and / or doxorubicin or a derivative thereof, preferably adriamycin, vincristine. 【0015】 According to a preferred embodiment, the amount of said molecule / drug ranges from 1 to 5 mg / ml, preferably the amount of paclitaxel (PTX - Taxol or Onxar) and its derivatives, preferably Abraxane, Docetaxel, to obtain anti-cancer effect / activity is 150 ng or more per 100 μl of said microfragmented adipose tissue or microfragmented lipoaspirate tissue and / or 300 ng or more per 100 μl of adipose tissue or lipoaspirate tissue sample. 【0016】 According to a preferred embodiment, the amount of said molecule / drug released per day ranges from 10-15% compared to the loading / priming amount of molecule / drug. 【0017】 A further aspect of the present invention relates to a delivery system comprising adipose tissue or a derivative thereof, preferably loaded with a molecule / or drug as disclosed above, for use in the treatment of cancer, wherein said cancer is preferably selected from: renal cell carcinoma, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mast cell tumor, lung cancer, breast adenocarcinoma, pancreatic adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer, more preferably selected from: pancreatic cancer, glioblastoma, neuroblastoma, and mesothelioma. 【0018】 drawing The invention is disclosed in more detail by way of non-limiting examples with reference to the following figures, in which: [Brief explanation of the drawings] 【0019】 [Figure 1] Figure 1 shows the in vitro inhibition of cancer cell proliferation, particularly the proliferation of pancreatic cancer cells (CFPAC1), following increasing doses of LPG / PTX and LASP / PTX. Tumor cell proliferation was assessed by crystal violet (CV) staining. Figure 1B shows the absorbance of CV eluted with acetic acid and measured at 550 nm. Figure 1C shows the anticancer activity tested using the MTT assay. The results show that increasing doses (25, 50, 100, and 200 μl) of LPG / PTX or LASP / PTX induced inhibition of cell proliferation. [Figure 2]Figure 2 shows the biological dosage of PTX released into the medium by various amounts of LPG / PTX (A) and LASP / PTX (B) (0.78–100 μl). The dosage was assessed by using an MTT assay to measure PTX activity on CFPAC1 cells. Table C reports the V50 values (volume that inhibits cell growth by 50%) for various amounts of LPG / PTX and LASP / PTX. [Figure 3] Figure 3 shows the PTX release kinetics for various doses of LPG / PTX and LASP / PTX. Histogram (A) reports the total paclitaxel equivalent concentration (p-EC) values for LPG / PTX and LASP / PTX. The R2 values represent the correlation coefficient of the dose-response kinetics. Table (B) reports the p-EC values alone and the PTX release as a percentage of the control (CTRL-100%). [Figure 4] Figure 4 shows that LPG / PTX and LASP / PTX inhibit the proliferation of primary GBM (GC-GBM) cancer cells. Photographs show control and untreated GC-GBM cells treated with a 50% dilution of SN obtained from LPG / PTX or LASP / PTX (added at 2 μg / ml). The results show the complete death of GC-GBM cells following the addition of LPG / PTX and LASP / PTX. SN obtained from control adipose tissue does not affect GC-GBM cell proliferation. There was no significant difference compared to the control medium (CTRL). Photographs were taken 72 hours after treatment. [Figure 5] Figure 5 shows that LPG / PTX and LASP / PTX cause IMR32 cell proliferation inhibition. Photographs show IMR32 cultured with or without a 50% dilution of SN obtained from LPG / PTX or LASP / PTX (added at 2 μg / ml), as well as control cultures. The results show that both LPG / PTX and LASP / PTX led to IMR32 cell death. Control CM obtained without added adipose tissue had no effect on IMR32 cell proliferation. Photographs were taken 72 hours after treatment. [Figure 6]Figure 6 shows that both LPG / PTX and LASP / PTX release drugs in a dose-dependent manner, causing long-term inhibition of IMR32 growth and, in addition, inhibiting angiogenesis. Figure 6A shows the dose-dependent activity of LPG / PTX and LASP / PTX. The results show that priming with 100 μl of LPG containing 300 ng of PTX is sufficient to prevent IMR32 growth. Figure 6B shows the sustained antitumor activity of LPG / PTX and LASP / PTX (even after 4 weeks). Figure 6C shows that SNs obtained from both LPG / PTX and LASP / PTX can inhibit endothelial cell (HUVEC) proliferation. [Figure 7] Figure 7 shows that freezing LPG / PTX did not affect its antiangiogenic and antitumor activity. After priming with PTX, LPG was kept at -20°C for 2 weeks, then thawed and tested. Figure 7A shows the antiangiogenic activity of SN obtained from LPG / PTX (tested at various dilutions in HUVECs) before and after freezing. Figure 7B shows the antitumor activity tested in GC-GBM cells. [Figure 8] Figure 8 shows the time-dependent release kinetics of PTX from both LPG / PTX and LASP / PTX. Anticancer activity against CFPAC1 cells was assessed by MTT assay, which measures the drug released into the SN (supernatant) from both LPG / PTX (A) and LASP / PTX (B) on days 1, 2, 5, and 7 of incubation. [Figure 9] Figure 9 shows the percentage of PTX release. The graph and table show the PTX release expressed as a percentage of the total amount (p-EC) and CTRL by both LPG / PTX (A) and LASP / PTX (B) on different days. P-EC represents the PTX equivalent concentration evaluated in the biological dosage assay. [Figure 10] - Figure 10 shows the biological dosage of bound / unbound PTX. Amount of bound / unbound PTX 5 minutes, 3 days and 6 days after treatment. Dose-response kinetics was used to calculate the values reported in Tables A and B. [Figure 11] Figure 11 shows photographs of LPG treated with 2 μg / ml of fluorescent PTX (PTX-F35) for 1 hour or 24 hours. The results show that 1 hour is sufficient for complete uptake of PTX-F35 by LPG. The results also show that PTX-F35 is primarily localized in the cytoplasm of LPG adipocytes. [Figure 12] Figure 12 shows the uptake and release of doxorubicin (DXR) by both LPG and LASP. The graph reports the biological activity of SN (supernatants) from both LPG (A) and LASP (B) treated with doxorubicin (DXR). SN were collected by washing LPG / DXR and LASP / DXR after 3 and 6 days of treatment and tested on CFPAC-1 cells. [Figure 13] Figure 13 shows the time-dependent release kinetics of DXR by both LPG / DXR and LASP / DXR. The histograms report the amount of DXR released by both LPG and LASP on days 3 and 6 after treatment, expressed as a percentage of the amount used to treat LPG and LASP (A) and as the total DXR equivalent concentration (d-EC) (B). Table (C) summarizes the values. DETAILED DESCRIPTION OF THE INVENTION 【0020】 Detailed Description of the Invention A first object of the present invention refers to a system for delivering molecules, preferably drugs, said delivery system comprising isolated adipose tissue or a derivative thereof. In other words, a first object of the present invention refers to a tissue-based system for delivering molecules, preferably drugs, wherein said tissue is isolated adipose tissue or a derivative thereof. 【0021】 Alternatively, the tissue-based system of the present invention can also be defined as a scaffold for loading molecules / drugs or a scaffold for delivering molecules / drugs. According to a preferred embodiment, the adipose tissue is fragmented, preferably microfragmented, as discussed in detail below. 【0022】 Therefore, in the context of the present invention, isolated adipose tissue, preferably microfragmented adipose tissue, is used as a scaffold (delivery system) for delivering large amounts of molecules, preferably drugs, more preferably lipophilic molecules and / or drugs, such as taxol (PTX) or any derivative thereof, or hydrophilic molecules and / or drugs, such as doxorubicin or any derivative thereof. 【0023】 The tissue-based system allows for the delivery of molecules and / or drugs to any part of the body or to an individual (any animal) in need thereof. Thus, the tissue-based system of the present invention allows for the administration of molecules and / or drugs in an individual (any animal) in need thereof. Preferably, the molecules / drugs are delivered to a site of interest, preferably a diseased and / or injured site. 【0024】 In the context of the present invention, fat tissue means adipose tissue. Preferably, said adipose tissue is isolated from any animal, more preferably it is isolated from a human, said human being being alive or dead. Preferably, said adipose tissue is derived from or isolated (purified) from any part of the body, preferably from the abdominal region and / or flank region. 【0025】 Preferably, the adipose tissue is isolated from the body by lipoaspiration / liposuction procedure (lipoaspirate), so according to a preferred embodiment, the adipose tissue is lipoaspirate (referred to as LASP in the examples and figures as an example of adipose tissue) or a derivative thereof. In the context of the present invention, liposuction, liposuction or simply lipo generally refers to the removal of adipose tissue (lipids) under negative pressure conditions by using a cannula. 【0026】 As mentioned above, adipose tissue, preferably lipoaspirate tissue, is microfragmented (LPG is used in the examples and figures as an example of microfragmented adipose tissue). Preferably, the adipose tissue is microfragmented by a non-enzymatic procedure, and therefore the lipids of the present invention are more preferably non-enzymatically microfragmented lipids. In other words, the lipids used or administered in the present invention as a delivery system are microfragmented without any enzymatic treatment. 【0027】 According to a further preferred embodiment of the present invention, the microfragmented adipose tissue is more preferably obtained by using a Lipogems® device (LPG) according to the procedure fully disclosed in patent application WO2011 / 145075. Adipose tissue, preferably lipoaspirate tissue, is introduced into the Lipogems® device, where it is gradually reduced (fragmented) into small clusters of adipose tissue, preferably by gentle mechanical force and more preferably in the presence of a solution, preferably a physiological saline solution. 【0028】 According to a preferred embodiment, the microfragmented lipid of the present invention preferably comprises clusters of adipose tissue having sizes ranging from 10 to 5000 μm, more preferably from 100 to 3000 μm, even more preferably from 200 to 2500 μm, more preferably from 300 to 1500 μm, more preferably from 400 to 900 μm. 【0029】 According to a further preferred embodiment, the lipid, preferably the microfragmented lipid or microfragmented lipid clusters, contain mesenchymal stem cells (MSCs), adipose-derived stem cells (ASCs), adipose stem cells, pericytes, adipocytes, and / or endothelial cells. In this regard, microfragmented lipid clusters are particularly advantageous because they maintain a natural / intact stromal vascular niche for resident cells, supported by a stroma that resembles the natural / physiological situation in terms of nutrition and / or signaling. Furthermore, the stroma provides a protective environment during cell grafting against any physical and / or chemical damage, e.g., mechanical, oxygen, etc. 【0030】 As such, the microfragmented lipids of the present invention are preferably characterized by: preferably 10 to 5000 μm, more preferably 100 to 3000 μm, Even more preferably, clusters of tissue having a size ranging from 200 to 2500 μm, even more preferably from 300 to 1500 μm, even more preferably from 400 to 900 μm; and / or 【0031】 - mesenchymal stem cells (MSCs), and / or adipose-derived stem cells (ASCs), and / or adipose stem cells, and / or pericytes, and / or adipocytes, and / or endothelial cells; and / or - Free of blood residue and / or pro-inflammatory oily substances. 【0032】 According to a preferred embodiment, the adipose tissue, preferably microfragmented adipose tissue or microfragmented adipose tissue, more preferably the resident cells, preferably mesenchymal stem cells (MSCs), and / or adipose-derived stem cells (ASCs), and / or adipose stem cells, and / or pericytes, and / or adipocytes, and / or endothelial cells, express at least one, preferably all, of the markers selected from: CD44, CD73, CD90, CD105, CD146, CD166 and any combination thereof; and / or at least one, more preferably all, of the following markers selected from: OCT4, SOX2, NANOG, b-tubulin III NESTIN, NEUROD1, MUSASHI1, PAX6, SOX3, and any combination thereof. 【0033】 More preferably, the cells, preferably said mesenchymal stem cells (MSCs), and / or adipose-derived stem cells (ASCs), and / or adipose stem cells, co-express the following panel of markers (signature characteristics): nestin, b-tubulin III, GFAP, and O4. Fat fragmentation within the device is preferably controlled by using one or more fragmentation / dispersion / emulsification means. 【0034】 According to a preferred embodiment, said means are metallic means, more preferably metallic beads and / or filters / nets, wherein said filters / nets preferably provide for micro-fragmentation of the tissue sample while the beads are free to move inside the device to promote separation between the solid and liquid parts of the tissue sample and (essentially) provide an emulsion of the liquid part with the washing fluid. 【0035】 Preferably, the beads have a size (average diameter) ranging from 0.1 to 30 mm, more preferably from 1 to 20 mm, even more preferably from 5 to 10 mm, even more preferably from 7.5 to 8.5 mm, and / or the filter / net has an average diameter ranging from 2000 μm to 200 μm, preferably from 1500 μm to 500 μm. The average mesh diameter of the filter / net ranges from 50 μm to 6000 μm, preferably from 500 μm to 3000 μm. 【0036】 More preferably, controlled shaking of the device is used to effect gentle movement of the fragmenting / disintegrating / emulsifying means throughout the adipose tissue. 【0037】 According to a preferred embodiment, fragmentation / dispersal / emulsification is preferably performed by immersion with a continuous flow of saline buffer through the device, thus allowing for easy washing of the tissue sample (specifically, effective removal of oily and / or bloody residues). More preferably, fragmentation / dispersal / emulsification is performed by washing the tissue sample with a continuous flow of saline buffer accompanied by bead shaking, which allows solid materials to lift towards the saline buffer inlet, and oily materials and / or bloody residues to flow out together with the saline towards the outlet. The fragmentation / disintegration / emulsification procedure preferably lasts for a few seconds. 【0038】 Therefore, according to a further preferred embodiment, the microfragmented lipids of the present invention are obtained by using gentle, enzyme-free, sterile, intraoperative and rapid procedures. 【0039】 The adipose tissue of the present invention is preferably isolated from any animal, more preferably from a human, preferably said animal / human being is healthy or cadaveric. According to a preferred embodiment, the lipids are isolated / adipose extracts from animal adipose tissue, more preferably human adipose tissue, more preferably from the abdominal and / or flank region of an individual, however, said lipids may be isolated from any useful body region. Preferably, the microfragmented lipids are autologous or xenogeneic. 【0040】 In the context of the present invention, the molecule to be delivered refers to any molecule, substance, or compound with biological and / or pharmacological activity, and / or at least one drug and / or prodrug or therapeutic agent. Preferably, the molecule is lipophilic (poorly water-soluble or water-insoluble). However, the tissue-based system of the present invention is also suitable for delivering hydrophilic molecules / drugs. 【0041】 For the purposes of the present invention, preferred molecules to be delivered are selected from: anti-inflammatory molecules, antibiotics, anti-cancer molecules, and 5α-reductase inhibitors (5-ARIs). 【0042】 The anti-cancer molecule (chemotherapeutic agent) is preferably selected from: natural products, preferably vinca alkaloids, more preferably: vinblastine, vincristine, and vinorelbine, taxanes, preferably paclitaxel or docetaxel, vincristine, vinblastine, nocodazole, epothilone and navelbine, epidipodophyllotoxin (teniposide), actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hepatic steroids, ... The compounds are selected from the group consisting of oxamethylmelamine oxaliplatin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16), adriamycin, amsacrine, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, pemetrexed, 5-FU, rafenib, methotrexate, cyclophosphamide, bortezomib, tomozolomide, sorafenib. Any combination of the previously reported molecules should be considered to form part of the present disclosure. 【0043】 More preferably, the molecule is selected from: paclitaxel (PTX - Taxol or Onxar) or a derivative thereof, Abraxane, and / or docetaxel, doxorubicin or a derivative thereof, preferably adriamycin and / or vincristine and any combination thereof. 【0044】 The anti-cancer molecule may likewise be delivered in combination with an additional molecule preferably selected from: antibiotics, anti-inflammatory substances, polyclonal or monoclonal antibodies, immunomodulatory molecules, biologics and combinations thereof. 【0045】 The molecule and / or drug / prodrug may be modified in any way, for example, pegylated, or it may be associated with a particle, preferably a nanoparticle, such as, for example, an albumin nanoparticle. 【0046】 According to a further aspect of the present invention, the tissue-based delivery system of the present invention, preferably loaded / primed with the molecules and / or drugs disclosed above, is used in the treatment of cancer. In the context of the present invention, the term "cancer" refers to any neoplastic disease, including cellular disorders such as, for example, renal cell carcinoma, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mast cell tumor, lung cancer, breast adenocarcinoma, pancreatic adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer. 【0047】 The most preferred types of cancer to be treated by using the delivery system of the present invention are selected from: pancreatic cancer, glioblastoma, neuroblastoma, mesothelioma, ovarian carcinoma, prostate cancer, and breast adenocarcinoma. 【0048】 Alternatively, the delivery system of the present invention, preferably loaded / primed with at least one molecule and / or drug as disclosed above, is used for the treatment of any disease or condition associated with or caused by an altered and / or enhanced proliferative state, preferably a hyperproliferative disease / disorder. 【0049】 The "growth state" of a cell refers to the rate of cell proliferation and / or state of cell differentiation. As used herein, "hyperproliferative disease / disorder" refers to any disorder that is caused by or manifests as unwanted cell proliferation in a patient. Preferably, the hyperproliferative disorder is selected from: psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, and abnormal wound healing, or neurodegenerative diseases, preferably amyotrophic lateral sclerosis, spinal muscular atrophy, multiple sclerosis, and traumatic nerve injury, such as spinal cord injury. 【0050】 As used herein, "proliferative" and "proliferation" refer to cells undergoing mitosis. According to a preferred embodiment, the amount of said molecule / drug that can be added / primed into the delivery system of the present invention ranges from 1-5 mg / ml adipose tissue. 【0051】 According to further preferred embodiments, the amount of paclitaxel (PTX - Taxol or Onxal) or its derivatives, preferably Abraxane, Docetaxel, to obtain anti-cancer effect / activity is 150 ng or more per 100 μl of microfragmented adipose tissue / lipoaspirate (LPG) and / or 300 ng or more per 100 μl of adipose tissue / lipoaspirate (LASP). 【0052】 Nevertheless, the maximum amount that can be added depends on the lipophilic-hydrophilic properties of the drug. The amount of molecule / drug released daily by the delivery system of the present invention ranges from 10-15% compared to the loading / priming amount, which is the amount used to prime microfragmented adipose tissue / lipoaspirate (LPG) and / or adipose tissue / lipoaspirate (LASP). 【0053】 A further aspect of the present invention refers to the delivery system of the present invention, preferably loaded with at least one molecule and / or drug as disclosed above, for use in the treatment of diseases or conditions caused by or associated with impaired (altered) angiogenesis, thus treating pathological angiogenesis. 【0054】 Besides cancer, in the context of the present invention diseases / conditions associated with or caused by altered angiogenesis refer to further diseases such as, for example, diabetic retinopathy or neuropathy. According to preferred embodiments of the present invention, the tissue-based delivery system is for topical, parenteral, peritoneal, mucosal, cutaneous, epithelial, subcutaneous, transdermal, intramuscular, nasal, oral, topical, vaginal, rectal or ocular administration. 【0055】 According to a further preferred embodiment, the tissue-based delivery system of the present invention finally loaded with the molecules / drugs disclosed above is administered / applied in combination with (pre-post) radiation therapy and / or surgery. Preferably, the tissue-based delivery system of the present invention finally loaded with the molecules / drugs disclosed above is applied to the involved area before surgery, e.g., to reduce the tumor area to be removed, thereby making the surgery less traumatic, especially for specialized areas such as the brain / eastern region. 【0056】 The tissue-based delivery system of the present invention finally loaded with the molecules / drugs disclosed above is preferably administered / applied before and / or after surgery, preferably topically, intraperitoneally, subcutaneously, preferably for preventing cancer recurrence, more preferably for metastatic tumors. [Example] 【0057】 Sample collection The results below refer to both the lipoaspirate tissue itself (LASP) and the Lipogems® tissue (LPG, ie, lipoaspirate tissue after processing with the Lipogems device - microfragmented lipoaspirate tissue). 【0058】 LASP was obtained by fat aspiration of the subcutaneous tissue as previously described (WO2011 / 145075) by using the disposable cannula provided with the Lipogems® kit. To obtain microfragmented adipose tissue -LPG-, LASP was processed by the Lipogems® device according to Bianchi et al., 2013 and Tremolada et al., 2016. 【0059】 drugs LPG and LASP were contacted with the following example chemotherapeutic agents: 1) Paclitaxel (PTX-stock solution 6mg / ml); 2) doxorubicin hydrochloride, and 3) Vincristine (VC) (stock solution 5 mg / ml). PTX, DXR and VC were diluted in culture medium at working / required concentrations as reported below. 【0060】 Priming Lipogems® and Lipoaspirate with Drugs Both LPG and LASP samples were vortexed for 1 min in 15 ml conical tubes (Euroclone, UK), followed by addition of PTX, DXR, and VC to a final concentration of 2 μg / ml. 【0061】 Samples LPG and LASP were vortexed for 1 minute and then incubated for 5 minutes or 24 hours at 37°C, 5% CO. After incubation, samples were mixed with 1 volume of Iscove's complete medium (IMDM + 10% FBS + 2 mM L-glutamine; Euroclone, UK), vortexed for an additional 1 minute, and centrifuged at 2500 × G for 10 minutes. 【0062】 The hydrophilic phase was immediately collected and replaced. This procedure was repeated various times as indicated in each experiment. Next, PTX, DXR, and VC-primed samples (LPG / PTX, LPG / DXR, LASP / PTX, LASP / DXR, LPG / VC, and LASP / VC) were processed according to various methodologies and tested for drug release. 【0063】 cell line The anticancer activity of PTX, DXR and VC was tested against the following cells: - Human pancreatic adenocarcinoma cell line CFPAC-1, cultured and grown in Iscove complete medium; 【0064】 - Primary glioblastoma (GBM) cell lines, also known as GC-GBM, cultured and grown in Neurocult medium (NC); and - The following neuroblastoma (NB) cell lines kindly donated by Dr. Mirco Ponzoni (Gaslini Hospital Genova, Italy): IMR32, HTLA230, SY5Y, SY5Y-LUC, NB1691 and NB1691-Luc. 【0065】 SY5Y-Luc, NB1691 and NB1691-Luc cells were cultured in RPMI1640 + 10% FCS. IMR32, HTLA230 and SY5Y cells were cultured in DMEM complete medium (Euroclone, UK) and passaged every 72 hours at a split ratio of 1:5. 【0066】 The anti-angiogenic activity of LPG and LASP added together with drugs was assayed in a human endothelial cell line (HUVEC). HUVECs were cultured in EGM complete medium (Lonza) and passaged 1:3 every week. 【0067】 Anticancer activity of LPG / PTX and LASP / PTX analyzed by transwell inserts Increasing volumes (25, 50, 100, 200 μl) of LPG / PTX and LASP / PTX were plated in 24-well plates (BD Falcon, USA, 1.9 cm diameter) using complete IMDM medium to a final volume of 700 μl. 2 ) were transfected with 2 × 10 cells in 300 μl of medium. 3 CFPAC-1 cells were seeded onto the upper insert (0.4 μm pore size; BD Falcon, USA). 【0068】 The effect of drugs released by LPG / LASP on tumor cell proliferation was assessed by staining adherent cells with 0.25% crystal violet (Sigma Aldrich, USA) for 10 min, followed by lysing the cells with 0.5 mL of 33% glacial acetic acid. The optical density (OD) of the eluted dye was measured at 550 nm (ChroMate, Awareness technology Inc, USA). The medium of the inserts was collected and tested for anticancer activity in a standardized biological dosing procedure to estimate the paclitaxel equivalent concentration (p-EC) according to the MTT assay. 【0069】 In some experiments, cells were detached and counted to assess their numbers. Results are expressed as the percentage of growth inhibition relative to control cells (CTRL) cultured in the absence of treatment. 【0070】 Anticancer activity of biological doses by MTT assay Supernatants (SN) from drug-primed LPG and LASP were assessed for CFPAC-1 cell proliferation ( Mosmann, 1983 ) by MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium; Sigma-Aldrich, USA). The inhibitory concentration (IC50) was determined according to the Reed and Muench formula (1938). 【0071】 According to the biological dosage system, the antitumor activity of LPG and LASP primed with PTX and DXR was compared with that of drug-free LPG and LASP and expressed as PTX or DXR equivalent concentrations (p-EC and d-EC, respectively), applying the following algorithm: p- or d-EC (ng / ml) = IC50 PTX or DXR (ng / ml) × 100 / V50-SN; where V50-SN is the volume of SN from primed LPG or LASP at which 50% inhibition is observed, and IC50 PTX or DXR is the concentration (ng / ml) of pure PTX or DXR that produces 50% inhibition. 【0072】 To assess the percentage of PTX or DXR released by priming LPG or LASP (% drug release), we calculated the total amount of drug released (p-EC or d-EC × volume of SN) related to the total amount of drug (μg) used to prime LPG or LASP. 【0073】 Time-dependent drug release kinetics from LPG / PTX and LASP / PTX LPG / PTX and LASP / PTX were diluted in equal volumes of Iscove's complete medium (Euroclone, UK). 【0074】 Samples were vortexed for 1 minute and then incubated at 37°C, 5% CO2 and for various incubation periods (1, 2, 3, 5, and 7 days). Culture supernatants (SN) were collected and tested for their anti-cancer activity in vitro. Supernatants from unprimed LPG or LASP were used as controls. 【0075】 Data details Results are expressed as mean ± standard deviation (SD). When necessary, differences between mean values were evaluated according to the Student's t-test performed by the GraphPad InStat program (GraphPad Software Inc., San Diego, CA, USA). A p-value ≤ 0.05 was considered statistically significant. Dose-response kinetics was analyzed using linear regression and by evaluating the correlation coefficient (R2) with Excel 2007 software (Microsoft, Inc.). 【0076】 result Inhibition of cancer growth in vitro by LPG / PTX and LASP / PTX The results show that increasing amounts (25, 50, 100, and 200 μl) of priming LPG (LPG / PTX) or LASP (LASP / PTX) resulted in a dramatic and complete inhibition of CFPAC-1 proliferation (FIG. 1A). 【0077】 The observed inhibition was greatest even in the lowest volume sample (Fig. 1B), and the amount of PTX released into 1 ml of medium by 25 μl of priming sample reached the IC concentration. Therefore, to estimate the PTX equivalent concentration (p-EC) in the transwells, we performed biological doses of 25, 50, 100, and 200 μl of medium plus LPG / PTX or LASP / PTX mixed in a final volume of 1 ml of medium (corresponding to dilutions of 1:40, 1:20, 1:10, and 1:5). 【0078】 The results show that the medium (hydrophilic) produces a dose-response inhibition accompanied by very high anticancer activity, with V50 values (volume that inhibits 50% of cell growth) reported in the boxes (Figure 2). The calculation of the total PEC values (Figure 3) confirmed that the PTX release was dependent on the amount of LPG or LASP priming sample. In fact, the results show that there is a significant correlation, well represented by R values resulting in 0.86 and 0.75 for LPG / PTX and LASP / PTX, respectively. 【0079】 Similar results were obtained by testing LPG / PTX and LASP / PTX on the CGBM and NB cell lines, IMR32. Specifically, the results in these cells show that the addition of various dilutions of SN derived from LPG / PTX and LASP / PTX to GC-GBM and IMR32 cell cultures resulted in potent dose-dependent growth inhibition. Furthermore, GC-GBM and IMR32 cells were treated with LPG / PTX or LASP / PTX-derived SN obtained after various incubation times. At each incubation time, the SN was aspirated and replaced with fresh medium. In these experiments, the durability of the antitumor effect was assessed. The results show that the anticancer effect of LPG / PTX on GC-GBM and IMR32, respectively, was maintained even after 4 weeks of incubation. 【0080】 In particular, the activity of LASP / PTX decreased more rapidly. Additionally, we performed experiments with PTX to determine the dose of drug sufficient to add to 100 μl of LPG or LASP. 【0081】 The results indicate that the minimum amount of PTX required to obtain an effective anticancer effect was approximately 150 ng per 100 μl of LPG and 300 ng per 100 μl of LASP for both tumor cell lines. The morphological appearance of GC-GBM and IMR32 cancer cells upon 72 h treatment with LPG / PTX- and LASP / PTX-derived SN and control lipid SN is shown in Figures 4 and 5 . The ability of LPG and LASP supplemented with PTX, DXR and VC to inhibit cancer cell proliferation was confirmed by using other NB tumor cell lines. 【0082】 Anti-angiogenic activity of LPG and LASP supplemented with anticancer drugs To evaluate the ability of LPG / PTX and LASP / PTX to affect angiogenesis, HUVECs were used. The previously described experiments on cancer cell lines were repeated using HUVECs (Figure 6). 【0083】 The results show that SNs derived from LPG / PTX and LASP / PTX can inhibit HUVEC proliferation in a dose-dependent manner, whereas SNs from untreated control LPG and LASP did not affect HUVEC proliferation (Figure 6A). The results indicate that the antiangiogenic effect of LPG / PTX is indeed strong and remained effective for up to 4 weeks, as observed for cancer cells. In particular, LASP / PTX shows a lower effect compared to the LPG / PTX one (Figure 6B). 【0084】 Interestingly, the amount of PTX required to induce an antiangiogenic effect was less than 150 ng per 100 μl of LPG; instead, 600 ng of PTX was required to have similar antiangiogenic activity with LASP (Figure 6C). Morphological analysis of HUVECs exposed to LPG / PTX or LASP / PTX showed strong nuclear fragmentation of the cells, suggesting that the mechanism of LPG / PTX or LASP / PTX on inhibiting HUVEC proliferation is probably related to cell apoptosis. 【0085】 Similar results were seen by using VC instead of PTX. Thawing and freezing did not affect the ability of LPG to load and release drug. In practice, LPG samples were kept at -20°C for 1 week, then thawed and treated with PTX, or fresh LPG samples were treated with PTX, frozen, kept at -20°C for 1 week, then thawed and tested for antitumor and antiangiogenic activity. 【0086】 LPG samples frozen either before or after treatment with PTX maintained their antitumor and antiangiogenic activity (Figure 7). The same results were observed when LPG was supplemented with DXR and VC or when LASP was used. However, in this case, a significant decrease in antitumor effect was observed when the drug was added at the time of LASP thawing. 【0087】 Time-dependent drug release kinetics from LPG / PTX and LASP / PTX To assess the amount of PTX released by primed LPG and LASP, a standard macro system in a tube was used. The test was set up by priming a 2 ml sample containing 2000 ng / ml PTX for 5 hours and then mixing it directly with an equal volume (1:2) of culture medium. 【0088】 This allowed for the confirmation of efficacy after a short priming period (5 hours), followed by a one-week release period. Samples were incubated at 37°C, and the medium (hydrophilic fraction) was collected on days 1, 3, 5, and 7 after centrifugation of the samples and replaced with fresh medium. The medium was collected on various days to assess their biological activity against cancer cells. The results demonstrate high activity up to the last day of culture tested (Figure 8). 【0089】 Estimation of percentage release by LPG / PTX and LASP / PTX The biological assay of PTX anticancer activity allowed us to estimate the amount of drug released into the medium per day for p-ECs. Release refers to the amount of PTX used to prime LPG or LASP (5,000 ng) and is expressed as the percentage of drug released and as the kinetics of drug accumulation (Figure 9). 【0090】 The results show that the total amount of PTX released on day 1 is 60.9 ng for LPG / PTX and 124.8 ng for LASP / PTX. The medium was replaced at each time point, so that we could estimate the kinetics of drug accumulation by integrating the amount of PTX released at each time point. On day 7, it was 175.7 ng for LPG / PTX and 302.7 ng for LASP / PTX. 【0091】 Moreover, the results show a decrease in the percentage of PTX released over time. Given this observation, to more fully understand the drug release kinetics, the possible correlation between the drug release percentage and sample dilution was evaluated. 【0092】 The results show a significant correlation between the relative drug release percentage and increasing sample dilution (this is more evident for LPG / PTX, R2 = 0.91, than for LASP / PTX, R2 = 0.52). This observation may be explained by the lipophilic chemical structure of PTX, which can be easily eluted from (and tightly incorporated into) LPG or LASP at higher dilutions of the hydrophilic medium. The higher the dilution, the greater the amount of serum albumin, a molecule with significant PTX affinity. 【0093】 These data demonstrate that LPG / LASP can release pharmacologically effective amounts of drug, in this case PTX, specifically effective anticancer activity for the tested drug, throughout the test day. As a result, LPG and / or LASP can be used to release effective amounts (very high dosages) of drug in the tumor area for several days, specifically by in situ injection. 【0094】 Moreover, we can also argue that the efficiency of in vitro release may be enhanced by the biology of the surrounding tissue into which LPG / PTX or LASP / PTX is injected (e.g., if that environment is more or less lipophilic). In any case, LPG or LASP used to release the drug into the tumor environment may reduce the systemic toxicity caused by intravenous chemotherapy. 【0095】 Study of PTX binding to LPG and whole LASP To measure the time required for PTX to bind to LPG or LASP and the ratio of bound / unbound drug, experiments were conducted. Specifically, sample priming was performed at various time points (72, 24, 5 h, and 5 min). 【0096】 The results were the same at all time points, therefore only PTX binding at 5 min was reported. After priming, the samples were immediately washed with medium and centrifuged to collect the unbound fraction of PTX. The floating fraction was also cultured in culture medium to measure drug release after 3 and 6 days. 【0097】 The results confirm that LPG bound approximately 95% (and 90% for LASP) of the PTX. Furthermore, after 3 and 6 days, PTX was still released in amounts capable of exerting significant inhibition of tumor cell growth (Figure 10). 【0098】 Drug distribution in LPG was assessed by using fluorescent PTX (PTX-F35). Results show that PTX-F35 (2 μg) was mostly adsorbed to the lipid fraction of LPG within 1 h, and no free drug was detected (Figure 11). Results remain the same up to 24 h. LASP shows the same drug distribution, even if it appears uneven. 【0099】 Preliminary doxorubicin experiments To understand whether LPG and LASP would be good scaffolds (delivery systems) for non-lipophilic drugs, the same experiments were performed using the hydrophilic antitumor drug doxorubicin (DXR). 【0100】 The biological dosage of DXR released into the medium 3 and 6 days after its replacement clearly demonstrates that both LPG and LASP can bind and subsequently release DXR in amounts effective against tumor growth in vitro. 【0101】 Further data confirm that LPG appears to perform better with almost two-fold higher release on day 6 compared to that released on day 3 (Figures 12 and 13).

Claims

[Claim 1] A scaffold to which a drug has been added, wherein the scaffold is isolated adipose tissue, the isolated adipose tissue is a cluster of microfragmented lipids or microfragmented aspirated adipose tissue and / or microfragmented lipids / aspirated adipose tissue containing mesenchymal stem cells (MSCs), adipose-derived stem cells (ASCs), adipose stem cells, pericytes, adipocytes, and endothelial cells, and the drug is selected from anti-inflammatory molecules, antibiotics, anticancer molecules, and 5α-reductase inhibitors. [Claim 2] The scaffold according to claim 1, wherein the isolated adipose tissue comprises cells expressing CD44, CD73, CD90, CD105, CD146, and CD166. [Claim 3] The scaffold according to claim 2, wherein the cells express OCT4, SOX2, NANOG, β-tubulin III, nestin, NEUROD1, MUSASHI1, PAX6, and SOX3. [Claim 4] The scaffold according to claim 2 or 3, wherein the cells express nestin, β-tubulin III, GFAP, and O4. [Claim 5] The scaffold according to any one of claims 1 to 4, wherein the adipose tissue, preferably the microfragmented adipose tissue or microfragmented aspirated adipose tissue, is isolated from any animal, more preferably from a living or dead human. [Claim 6] The aforementioned anticancer molecule (chemotherapeutic agent) is selected from vinca alkaloids, more preferably vinblastine, vincristine, and vinorelbine, taxane, preferably paclitaxel or docetaxel, vincristine, vinblastine, nocodazole, epothilon and navelbine, epidipodophilotoxin (teniposide), actinomycin, amsacrin, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxane, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifospha A scaffold according to any one of claims 1 to 5, selected from mid, melphalan, merchloretamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16), adriamycin, amsacrin, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, pemetrexed, 5-FU, rafenib, methotrexate, cyclophosphamide, bortezomib, tomozolomide, sorafenib, and any combination thereof. [Claim 7] The scaffold according to claim 6, wherein the anticancer molecule is selected from paclitaxel (PTX-Taxol or Onxal), Abraxane, and / or docetaxel, doxorubicin, adriamycin, or vincristine, and any combination thereof. [Claim 8] The scaffold according to claim 6 or 7, wherein the anticancer molecule is selected from paclitaxel (PTX-Taxol or Onxal), doxorubicin, vincristine, and any combination thereof. [Claim 9] The scaffold according to any one of claims 1 to 8, wherein the amount of the drug is in the range of 1 to 5 mg / ml. [Claim 10] The scaffold according to claim 9, wherein the amount of paclitaxel (PTX-Taxol or Onxal), Abraxane, and docetaxel is 150 ng or more per 100 μl of the microfragmented adipose tissue or microfragmented aspirated adipose tissue, or 300 ng or more per 100 μl of adipose tissue / aspirated adipose tissue. [Claim 11] A scaffold to which the drug according to any one of claims 1 to 5 is added, The drug is an anticancer molecule according to any one of claims 6 to 10 for use in the treatment of cancer, a scaffold. [Claim 12] The scaffold for use according to claim 11, wherein the cancer is selected from renal cell carcinoma, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mast cell tumor, lung cancer, mammary gland adenocarcinoma, pancreatic adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or gastric cancer. [Claim 13] The scaffold for use according to claim 12, wherein the cancer is selected from pancreatic cancer, glioblastoma, neuroblastoma, and mesothelioma.

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