Patent Description:
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.

Drugs have long been used to improve health and extend lives.

It is well known that the delivering approach can influence significantly the efficacy of a drug. Indeed, some drugs are characterized by an optimum concentration range within which maximum benefit is derived, and concentrations out this range can be toxic or produce no therapeutic benefit at all.

On the other hand, the very slow progress in the efficacy of the treatment of diseases has increased the need for a multidisciplinary approach to the delivery of therapeutics to targets in tissues.

This need has caused a dramatic change in the practice of drug delivery in the past few decades and even greater changes are anticipated in the near future.

In particular, a distinct concept and delivery method has been recently developed that is based on the use of cells as therapeutic carriers. Cell-based vehicles are particularly attractive for delivery of bio-therapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction.

The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimizing inevitable adverse side effects, is an appealing perspective that can be applied in many clinical settings.

<CIT> relates to a product comprising a cellular carrier including physiological mesenchymal cells, and a cytoxic chemotherapeutic drug internalized by said physiological mesenchymal cells.

The present invention proposes as solution to the need reported above a delivery system made of fat tissue or derivatives thereof. In particular, the fat tissue is micro-fragmented fat tissue and/or micro-fragmented lipoaspirate.

Indeed, the authors of the present invention have surprisingly found that:.

In particular, the effects reported above, have been experimentally demonstrated by using both lipoaspirate as such and micro-fragmented lipoaspirate, that is the fat tissue obtained after the controlled micro-fragmentation process of the lipoaspirate performed as disclosed below.

However, the micro-fragmented fat delivery system is more advantageous than lipoaspirate (fat tissue) as such since it is easier to be handled and it allows a better standardization and reliability of the therapeutic results. In this regard, it is well known how important the standardization and reliability for any therapeutic use are.

A first aspect of the present invention refers to a scaffold loaded with drugs, wherein said scaffold is isolated fat tissue, wherein the isolated fat tissue is micro-fragmented fat or a micro-fragmented lipoaspirate and/or clusters of micro-fragmented fat/lipoaspirate comprising Mesenchymal Stem Cells (MSCs), Adipose-derived Stem Cells (ASCs), Adipose Stem Cells, pericytes, adipocytes and endothelial cells and wherein said drugs are selected from: antiinflammatory molecules, antibiotics, anti-cancer molecules, and 5a-Reductase inhibitors.

The tissue is micro-fragmented fat or micro-fragmented lipoaspirate, preferably isolated from any animal, more preferably it is isolated from humans said humans being alive or cadaver and/or preferably comprising clusters of micro-fragmented fat/lipoaspirate having size ranging preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>, still more preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>.

The anti-cancer molecules (chemotherapeutics) are preferably selected from: natural products, preferably vinca alkaloids, more preferably selected from: vinblastine, vincristine, and vinorelbine, taxane, preferably paclitaxel or docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (teniposide), actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16)), adriamycin, amsacrine, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-<NUM>) and mitoxantrone, pemetrexed, <NUM>-FU, rafenib, metotrexate, cyclophosphamide, bortezomib, tomozolomide, sorafenib. and any combination thereof.

More preferably, the anti-cancer molecules are selected from: Paclitaxel (PTX - Taxol or Onxal) or derivatives thereof, preferably Abraxane, Docetaxel and/or doxorubicin or derivative thereof, preferably Adriamycin, Vincristine.

According to a preferred embodiment, the amount of said molecules/drugs ranges from <NUM> to <NUM>/ml, preferably the amount of Paclitaxel (PTX - Taxol or Onxal) and derivatives thereof, preferably Abraxane, Docetaxel, for obtaining an anti-cancer effect/activity is not less than <NUM> ng for 100µl of said micro-fragmented fat tissue or micro-fragmented lipoaspirate and/or not less than <NUM> ng for 100µl of fat tissue or lipoaspirate sample.

According to a preferred embodiment, the amount of said molecules/drugs released per day ranges from <NUM>-<NUM> % compared to the loading/priming amount molecules/drugs.

A further aspect of the present invention, refers to the scaffold loaded with anti-cancer molecules/or drugs as disclosed before, for use in the treatment of a cancer, wherein said cancer is preferably selected from: renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, pancreatic adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer, more preferably selected from: pancreatic cancer, glioblastoma, neuroblastoma and mesotelioma.

The invention will be disclosed in more detail by means of non-limiting examples referring to the following figures.

A first object of the present invention refers to a scaffold loaded with drugs, wherein said scaffold is isolated fat tissue, wherein the isolated fat tissue is micro-fragmented fat or a micro-fragmented lipoaspirate and/or clusters of micro-fragmented fat/lipoaspirate comprising Mesenchymal Stem Cells (MSCs), Adipose-derived Stem Cells (ASCs), Adipose Stem Cells, pericytes, adipocytes and endothelial cells and wherein said drugs are selected from: antiinflammatory molecules, antibiotics, anti-cancer molecules, and 5a-Reductase inhibitors.

The tissue-based system allows the delivery of molecules and/or drugs in the body or any part of the body of an individual (any animal) in need thereof. Therefore, the tissue-based system of the invention allows molecules and/or drugs administration in individuals (any animal) in need thereof. Preferably, the molecules/drugs are delivered in the interested site, preferably the sick and/or the injured site.

In the contest of the present invention, fat tissue means adipose tissue. Preferably, said fat tissue is isolated from any animal, more preferably it is isolated from a humans said humans being alive or a cadaver.

Preferably, said fat tissue derives/is isolated (purified) from any part of the body, preferably from the lower and/or the lateral abdomen area. Preferably, said fat tissue is isolated from the body by lipoaspiration/liposuction (lipoaspirate) procedure. Therefore, according to a preferred embodiment the fat tissue is a lipoaspirate (LASP in the example and drawings as an example of fat tissue) or derivatives thereof. In the context of the present invention, lipoaspiration or liposuction or simply lipo means the removal of adipose tissue (fat) under negative pressure condition, generally by using a cannula.

As already mentioned before, the fat tissue, preferably the lipoaspirate, is micro-fragmented (LPG in the example and drawings as an example of micro-fragmented fat tissue). Preferably, the fat tissue is micro-fragmented by a non-enzymatic procedure and therefore the fat of the present invention is more preferably non-enzymatic micro-fragmented fat. In other words, the fat used/administered in the present invention as delivery system has been micro-fragmented without any enzymatic treatment.

According to a further preferred embodiment of the present invention, the micro-fragmented fat tissue is obtained by using the Lipogems® device (LPG), more preferably according to the procedure as fully disclosed in the <CIT>.

The fat tissue, preferably the lipoaspirate, is introduced in the Lipogems® device wherein it is progressively reduced (fragmented) in small clusters of fat tissue preferably by means of mild mechanical forces and, more preferably, in presence of a solution, preferably a saline solution. According to a preferred embodiment, the micro-fragmented fat of the invention contains clusters of fat tissue having size ranging preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>, still more preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>.

The micro-fragmented fat or the clusters of micro-fragmented fat, comprise 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. In this regard, particularly advantageous are the micro-fragmented fat clusters since they keep the natural/intact stromal vascular niche of the resident cells that, consequently, are supported by the stroma resembling the natural/physiological context in trophic and/or signaling terms. Additionally, the stroma provides a protected environment during the graft of the cells against any physical and/or chemical insults, such as mechanical, oxygen, ecc.

Therefore, the micro-fragmented fat of the present invention is preferably characterized by:.

According to a preferred embodiment, the isolated fat tissue comprises cells expressing at least one, preferably all, marker selected from: CD44, CD73, CD90, CD105, CD146 CD166 and any combination thereof; and/or at least one marker, more preferably all, selected from: OCT4, SOX2, NANOG, b-tubulin III NESTIN, NEUROD1, MUSASHI1, PAX6, SOX3 and any combination thereof.

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): nestin, b-tubulin III, GFAP, and O4.

Fat fragmentation inside the device is preferably controlled by using one or more fragmentation/disaggregation/emulsifying means.

According to a preferred embodiment, said means are metallic means, more preferably metallic beads and/or filters/nets, wherein the filters/nets provide preferably a micro-fragmentation of the tissue sample, while the beads freely move inside the device in order to promote the separation between the solid part and the liquid part of the tissue sample and (inherently) provide an emulsion of the liquid parts with the a washing fluid. Preferably the beads have size (average diameter) ranging preferably from <NUM>,<NUM>-<NUM> millimeters, more preferably <NUM>-<NUM>, still more preferably <NUM>-<NUM>, still more preferably <NUM>,<NUM>-<NUM>,<NUM> and/or said filter/nets have average diameter ranging from <NUM> to <NUM>, preferably from <NUM> to <NUM>.

The mesh average diameter (pore size) of the filter/net ranges between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

It is advisable to perform mild movements of the fragmentation/disaggregation/emulsifying means throughout the fat tissue, more preferably by performing a controlled shaking of the device.

According to a preferred embodiment, the fragmentation/disaggregation/emulsification is performed in immersion, preferably with a continuous flow of saline buffer through the device, so allowing an easy washing of the tissue sample (in particular an effective oil and/or blood residues removal). More preferably, the fragmentation/disaggregation/emulsification is performed by washing the tissue sample through a continuous flow of the saline buffer that, together with beads shaking, allows the solid material to lift towards the inlet of the saline buffer, leaving the oil and/or blood residues to flow together with the saline towards the outlet.

The fragmentation/disaggregation/emulsification procedure lasts for preferably few seconds.

Therefore, according to a further preferred embodiment, the micro-fragmented fat of the present invention is obtained by using a gentle, enzyme-free, sterile, intra-operative and rapid manipulation.

The fat tissue of the present invention is preferably isolated from any animal, more preferably from humans. Preferably said animal/human is healthy or cadaver.

According to a preferred embodiment, the fat is animal adipose tissue, more preferably human adipose tissue, more preferably isolated/lipoaspirate from the lower and/or the lateral abdomen area of an individual. However, said fat can be isolated from any useful body area.

Preferably, the micro-fragmented fat is autologous or heterologous.

For the purpose of the present invention, the molecules to be delivered are selected from: anti-inflammatory molecules, antibiotics, anti-cancer molecules, and 5α-Reductase inhibitors (<NUM>-ARIs). Said anti-cancer molecules (chemotherapeutics) are preferably selected from: natural products, preferably vinca alkaloids, more preferably selected from: vinblastine, vincristine, and vinorelbine, taxane, preferably paclitaxel or docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (teniposide), actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16)), adriamycin, amsacrine, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-<NUM>) and mitoxantrone, pemetrexed, <NUM>-FU, rafenib, metotrexate, cyclophosphamide, bortezomib, tomozolomide, sorafenib. Any combination of the molecules reported above should be considered forming part of the present disclosure.

More preferably, the molecules are selected from: Paclitaxel (PTX - Taxol or Onxal) or derivatives thereof, preferably selected from Abraxane and/or Docetaxel, doxorubicin or derivative thereof, preferably Adriamycin and/or, Vincristine and any combination thereof.

The anti-cancer molecules can be delivered also in combination with further molecules, preferably selected from: antibiotics, anti-inflammatory substances, poli- or mono-clonal antibodies, immunomodulatory molecules, biological drugs and combinations thereof.

The molecule and/or the drug/prodrug can be modified in any way, such as pegylation or it can be associated with particles, preferably nanoparticles, such as albumin-nanoparticles.

According to a further aspect of the present invention, the tissue-based delivery system of the invention, loaded/primed with anti-cancer molecules and/or drugs as disclosed above, is used for the treatment of a cancer.

In the context of the present invention the term "cancer" refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, pancreatic adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer.

The most preferred type of cancer to be treated by using the delivery system of the present invention is selected from: pancreatic cancer, glioblastoma, neuroblastoma, mesothelioma, ovarian carcinoma, and prostatic cancer, and mammary adenocarcinoma.

Alternatively, the delivery system of the invention, 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 increased growth state, preferably a hyperproliferative disease/disorder.

The "growth state" of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell.

As used herein, "hyperproliferative disease/disorder" refers to any disorder, which is caused by or is manifested by unwanted proliferation of cells in a patient. Preferably, hyperproliferative disorders are selected from: psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, and abnormal wound healing, or neuro degenerative diseases, preferably amyotrophic lateral sclerosis, spinal muscular atrophy, multiple sclerosis, and traumatic neural injury, such as spinal cord lesion.

As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis.

According to a preferred embodiment, the amount said molecules/drugs that can be loaded/primed into the delivering system of the invention ranges from <NUM> to <NUM>/ml of fat tissue.

According to a further preferred embodiment, the amount of Paclitaxel (PTX - Taxol or Onxal) or derivatives thereof, preferably Abraxane, Docetaxel, for obtaining an anti-cancer effect/activity is not less than <NUM> ng for 100ul of micro-fragmented fat tissue/lipoaspirate (LPG) and/or not less than <NUM> ng for 100ul of fat tissue/lipoaspirate(LASP).

Nevertheless, the amount that can be loaded as maximum depends on the lipo-hydrophylic nature of the drug.

The amount of the molecules/drugs released per day by the delivery system of the invention ranges from <NUM>-<NUM>% compared to the loading/priming amount that is the amount used to prime the micro-fragmented fat tissue/lipoaspirate (LPG) and/or fat tissue/lipoaspirate (LASP).

A further aspect of the present invention refers to the delivery system of the invention, loaded with at least one molecule and/or drug as disclosed above for use in the treatment of a disease or a condition caused by or associated with impaired (altered) angiogenesis, therefore for treating pathological angiogenesis.

Besides cancer, in the context of the present invention, for disease/condition associate with or caused by altered angiogenesis is meant further diseases such as diabetic retinopathy or neuropathy.

According to a preferred embodiment of the invention, the tissue-based delivery system is for local, parenteral, peritoneal, mucosal, dermal, epidermal, subcutaneous, transdermal, intramuscular, nasal, oral, topical, vaginal, rectal or intra-ocular administration.

According to a further preferred embodiment, the tissue-based delivery system of the invention, loaded with the molecules/drugs as disclosed above, is administered/applied in combination (pre-post) radiotherapy and/or surgery. Preferably the tissue-based delivery system of the invention, loaded with the molecules/drugs as disclosed above, is applied on the interested area before surgery for example in order to reduce the tumor area to be removed and therefore, to make the surgery less traumatic especially for specific area such as brain/head.

The tissue-based delivery system of the invention, loaded with the molecules/drugs as disclosed above, is preferably pre and/or post-operatory administration/application, preferably topical, intraperitoneal, subcutaneous, administration/application, preferably for preventing the cancer relapses, more preferably for metastatic tumors.

The following results refer to both lipoaspirate as such (LASP, not according to the invention) and Lipogems® tissue (LPG that is the lipoaspirate after treatment with Lipogems device - micro-fragmented lipoaspirate).

LASP has been obtained by liposuction of subcutaneous tissue as previously described (<CIT>) by using disposable cannulas provided with the Lipogems® kit.

In order to obtain micro-fragmented adipose tissue - the LPG -, the LASP was processed by the Lipogems® device according to Bianchi et al. , <NUM> and Tremolada et al.

LPG and LASP samples were exposed to the following example of chemotherapy drugs:.

PTX, DXR and VC are diluted in culture medium as reported below at the working/requested concentration.

Samples of both LPG and LASP were vortexed for <NUM> minute in <NUM> conical tube (Euroclone, UK). Subsequently, PTX, DXR and VC were added to the samples at a final concentration of <NUM>µg/ml.

The samples LPG and LASP were vortexed <NUM> minute and then incubated <NUM> minutes or <NUM> hours at <NUM>, <NUM>% CO2. After the incubation the samples were mixed with <NUM> volume of Iscove complete medium (IMDM +<NUM>% FBS + <NUM> L-glutamine; Euroclone, UK), further vortexed <NUM> minute and centrifuged at <NUM> xG, <NUM>.

The hydrophilic phase was immediately collected and replaced.

This operation was repeated at different times as described in each experiment.

The PTX, DXR, VC-primed samples (LPG/PTX, LPG/DXR, LASP/PTX, LASP/DXR, LPG/VC, LASP/VC) were then processed according to different methodology to study the drug release.

The anticancer activity of PTX, DXR and VC was tested on the following cells:.

SY5Y-Luc, NB1691 and NB1691-Luc cells were cultured in RPMI <NUM> + <NUM>%FCS.

IMR32, HTLA230 and SY5Y cells were cultured in DMEM complete medium ((Euroclone, UK) and passed every <NUM> at split ratio <NUM>:<NUM>.

The anti-angiogenic activity of the LPG and LASP loaded with drugs has been assayed on human endothelial cell line (HUVEC).

HUVECs were grown in EGM completed medium (Lonza) and passed weekly <NUM>:<NUM>.

Increasing volumes (<NUM>, <NUM>, <NUM>, <NUM>µl) of LPG/PTX and LASP/PTX were introduced in <NUM>-well plate (BD Falcon, USA, diameter <NUM> cm2) using complete IMDM medium to a final volume of <NUM>µl. <NUM>*<NUM> CFPAC-<NUM> cells in <NUM>µl of medium have been seeded into the upper insert (<NUM> pore size; BD Falcon, USA).

The effect of the drugs released by the LPG/LASP on the growth of the tumor cells has been evaluated by staining the adherent cells with <NUM>% crystal violet (Sigma Aldrich, USA) for <NUM> followed by cell lysing with <NUM> of <NUM>% glacial acetic acid.

The optical density (OD) of the eluted dye was measured at <NUM> (ChroMate, Awareness technology Inc, USA).

The medium of the inserts was collected to test the anticancer activity in a standardized biological dosage procedure to estimate the Paclitaxel equivalent concentration (p-EC) according to a MTT assay.

In some experiments, the cells were detached and counted to evaluate their number.

The results are expressed as percentage of growth inhibition referred to control cells growing in the absence of treatment (CTRL).

The supernatants (SN) from LPG and LASP primed with drugs were evaluated by MTT assay (<NUM>-(<NUM>,<NUM>-dimethyl-<NUM>-thiazolyl)-<NUM>,<NUM>-diphenyl-<NUM>-H-tetrazolium; Sigma-Aldrich, USA) on CFPAC-<NUM> cell proliferation (Mosmann, <NUM>).

The inhibitory concentration (IC50) was determined according to the Reed and Muench formula (<NUM>).

According to a biological dosage system, the anti-tumour activity of PTX and DXR primed LPG and LASP was compared to that of free drugs and expressed as PTX or DXR-equivalent concentration (respectively p-EC and d-EC), applying the following algorithm: p- or d-EC(ng/ml) = IC50 PTX or DXR (ng/ml)×<NUM>/ V50-SN; where the V50-SN is the volume of SN from primed LPG or LASP at which the <NUM>% of inhibition was observed and IC50 PTX or DXR is the concentration (ng/ml) of pure PTX or DXR producing <NUM>% of inhibition.

In order to evaluate the percentage of PTX or DXR released by the primed LPG or LASP (drug release %) we calculated the total amount of drug released (p-EC or d-EC x volume of SN) referred to the total amount (µg) of drug used to prime LPG or LASP.

LPG/PTX and LASP/PTX were diluted into equal volume of Iscove complete medium (Euroclone, UK). The samples were vortexed for one minute and then incubated at <NUM>, <NUM>%CO2 and at different times of incubation (<NUM>, <NUM>, <NUM>, <NUM> and <NUM> days). The medium supernatant (SN) was collected to test its anticancer activity in vitro. Supernatants from unprimed LPG or LASP were used as control.

The results are expressed as mean ± standard deviation (SD). Where requested, the differences between mean values were evaluated according to Student's t-test performed by GraphPad InStat program (GraphPad Software Inc. , San Diego, CA, USA). p-Values ≤ <NUM> were considered statistically significant. The dose-response kinetics were analysed by using linear regression and evaluating the correlation coefficient (R2) by Excel <NUM> software (Microsoft, Inc.

The results show that increasing amount (<NUM>, <NUM>, <NUM> and <NUM>µl) of primed LPG (LPG/PTX) or LASP (LASP/PTX) produced a dramatic and complete inhibition of the growth of CFPAC-<NUM> (<FIG>.

The observed inhibition is maximal also at the lowest amount of sample (<FIG> meaning that the amount of PTX released in <NUM> of medium by <NUM>µl of the primed sample reached a concentration of IC90. Therefore, in order to estimate the PTX equivalent concentration (p-EC) in the transwell, we performed a biological dosage of the culture medium receiving <NUM>, <NUM>, <NUM> and <NUM>µl of LPG/PTX or LASP/PTX that was mixed to a final volume of <NUM> of medium (corresponding to a dilution of <NUM>:<NUM>; <NUM>:<NUM>; <NUM>:<NUM> and <NUM>:<NUM>). The results show that the media (hydrophilic) produced a dose response inhibition with very high anticancer activity as indicated by the V50 value (volume inhibiting <NUM>% cell growth) reported in the box (<FIG>).

The calculation of total PEC values (<FIG>) confirmed that the releasing of PTX is dependent on the amount of primed samples of LPG or LASP. Indeed, the results show that there is a significant correlation well expresses by the R2 values that resulted of <NUM> and <NUM> for LPG/PTX and LASP/PTX respectively.

Similar results were obtained by testing LPG/PTX and LASP/PTX on CG-GBM cell line and on NB cell line IMR32. In particular, the results on these cells show that the addition of SN at different dilutions derived from LPG/PTX and LASP/PTX to culture of GC-GBM and IMR32 cells produced a potent dose dependent growth inhibition. Additionally, GC-GBM and IMR32 cells were treated with LPG/PTX or LASP/PTX derived SN, obtained after different time of incubation. At each time of incubation, SN was aspirated and replaced with fresh medium. These experiments were performed to assess the antitumor effect duration. The results show that the antitumor effect of LPG/PTX on GC-GBM and IMR32 respectively was maintained even after <NUM> weeks of incubation.

In particular, the activity of LASP/PTX decreased more rapidly.

Further, we performed experiments with PTX to establish the dose of drug sufficient to load <NUM>µl of LPG or LASP.

The results show that the minimal dose necessary of PTX to obtain an efficient antitumor effect was around <NUM> ng for 100ul of LPG and 300ng for 100ul of LASP on both tumor cell lines.

The morphological appearance of GC-GBM and IMR32 cancer cells upon <NUM> treatment with LPG/PTX and LASP/PTX derived SN and control fat SN is shown in <FIG>.

The capacity of LPG and LASP loaded with PTX, DXR and VC of inhibiting cancer cell proliferation was confirmed by using other NB tumor cell lines.

Anti-angiogenic activity of LPG and LASP loaded with anticancer drugs.

In order to evaluate the capacity of LPG/PTX and LASP/PTX to affect angiogenesis HUVECs were used. The experiments disclosed above referring to the cancer cell lines were repeated using HUVECs (<FIG>).

The results show that SNs derived from LPG/PTX and LASP/PTX are able to inhibit in a dose dependent manner the growth of HUVECs. On the other end, SNs from control untreated LPG and LASP did not affect the growth of HUVECs (<FIG>). The results show that the anti-angiogenic effect of LPG/PTX is really high and as observed for the cancer cells, it was maintained effective for up to <NUM> weeks.

In particular, LASP/PTX show a reduced effect compared to the one of LPG/PTX (<FIG>).

Interestingly, the amount of PTX needed to induce anti-angiogenic effects is less than 150ng for 100ul of LPG; instead, in order to have similar antiangiogenic activity with LASP, <NUM> ng of PTX was required (<FIG>). The morphological analysis of HUVECs upon exposure to LPG/PTX or LASP/PTX show a potent nuclear fragmentation of the cells suggesting that the mechanism of LPG/PTX or LASP/PTX on HUVECs growth inhibition is probably associated with cell apoptosis.

Similar results were seen by using VC instead of PTX.

Thawing and freezing did not affect LPG capacity to load and release drugs.

Indeed, samples of LPG were kept at -<NUM> for <NUM> week and then thawed and treated with PTX. Alternately, fresh LPG samples were treated with PTX and frozen, maintained at -<NUM> for <NUM> week and then thawed to test antitumor and anti-angiogenic activity.

LPG samples, frozen either before or after the treatment with PTX, keep their antitumor and anti-angiogenic activities (<FIG>).

The same results were observed when LPG was loaded with DXR and VC or when LASP was used.

However, in this case, a significant decrease of the antitumor efficacy was observed if drug was loaded upon LASP thawing.

Time releasing kinetics of drug by LPG/PTX and LASP/PTX.

In order to evaluate the amounts of PTX released by the primed LPG and LASP a standard macro-system in tubes has been used.

The study was setup by priming <NUM> of sample with <NUM> ng/ml of PTX for <NUM> hours and then directly mixing it with the same volume (<NUM>:<NUM>) of culture medium.

This allowed verifying the efficacy of a short time of priming (5hs) and the subsequent release until one week. The sample was incubated at <NUM>° C and at day <NUM>, <NUM>, <NUM> and <NUM> after sample centrifugation, the medium (hydrophilic fraction) was collected and replaced with new medium. The media have been collected at the different days for evaluating their biological activity on cancer cells. The results demonstrate high activity until the last days of culture tested (<FIG>).

The biological assay of PTX anticancer activity, allowed estimating the amount of drug released in the medium per day in term of p-EC. The release is referred to the amount of PTX used to prime LPG or LASP (that is <NUM> ng) and is expressed as the percentage of drug released and as a kinetics of drug accumulation (<FIG>).

The results show that the total amount of PTX released at day <NUM> is <NUM> ng for LPG/PTX and <NUM> ng for LASP/PTX.

The medium has been replaced at each time. Therefore, we can estimate a kinetic of drug accumulation by integrating the amount of PTX released at each time. A day <NUM> it is of <NUM> ng for LPG/PTX and <NUM> ng for LASP/PTX.

Moreover, the results show a decrease of the percentage of the released PTX along the time.

In view of this observation, the possible correlation between the percentage of drug release and the dilution of the sample has been evaluated in order to better appreciate the drug releasing kinetics.

The results show a significant correlation between the percentage of drug release related and the increasing of the sample dilution (this is more evident for LPG/PTX, R2 =<NUM> than LASP/PTX R2=<NUM>). This observation could be explained by the lipophilic chemical structure of PTX that can be easier eluted from LPG or LASP (in which is strongly incorporated) at higher dilutions of the hydrophilic medium. The higher is the dilution the higher is the amount of serum albumin that is a molecule having a significant PTX affinity.

These data demonstrate that LPG/LASP is able to release all along the tested days a pharmacological effective amount of the drug, the PTX in this case, in particular for the tested drug, an effective anticancer activity. Therefore, LPG and/or LASP can be used to release an effective drug amount (very high dosage) in the tumour area for several days, in particular, by the in situ injection.

Moreover, we can also argue that the efficiency of release in vivo can be facilitate by the biological structure of the surrounding tissue into which LPG/PTX or LASP/PTX are injected (e.g. if the environment is more or less lipophilic). In any case, LPG or LASP used to release drug in the tumour environment could reduce the systemic toxicity that occurs by intravenously chemotherapy.

Experiments have been set up to measure the time the PTX needs to bind LPG or LASP and the rate of bound/unbound drug. In particular, the priming of the samples has been performed at different time (<NUM>, <NUM>, <NUM> hours and <NUM> minutes).

The results are the same at any time therefore, here has been reported only the binding of PTX at <NUM> minutes.

After priming, the samples were immediately washed with the medium and centrifuged to collect the unbound fraction of PTX.

The floating fraction was also cultured in the medium for measuring the drug release after <NUM> and <NUM> days.

The results confirm that LPG bound about <NUM>% of PTX (and <NUM>% for LASP). Furthermore, after three and six days, PTX is still released in an amount able to exert a significant inhibition of the tumour cell proliferation (<FIG>).

The distribution of the drug in LPG has been evaluated by using a fluorescent PTX (PTX-F35). The results show that PTX-F35 (2ug) is mostly adsorbed in the lipid fraction of LPG in one hour (<FIG>) and no free drug has been detected. The results are the same until 24hours. LASP shows the same drug distribution even if it appears less uniform.

Claim 1:
A scaffold loaded with drugs, wherein said scaffold is isolated fat tissue, wherein the isolated fat tissue is micro-fragmented fat or a micro-fragmented lipoaspirate and/or clusters of micro-fragmented fat/lipoaspirate comprising Mesenchymal Stem Cells (MSCs), Adipose-derived Stem Cells (ASCs), Adipose Stem Cells, pericytes, adipocytes and endothelial cells and wherein said drugs are selected from: anti-inflammatory molecules, antibiotics, anti-cancer molecules, and 5α-Reductase inhibitors.