Method and device for drug delivery

Abstract

This disclosure relates to methods of promoting infiltration of a nanoparticle-based anti-cancer drug into a solid tumor of a subject using mechanical stimulation. Devices for performing such a method are also disclosed. In one embodiment, the mechanical stimulation is a compressive force applied to the tumor or to a tissue surrounding the tumor, or to a tissue in the vicinity of the tumor. In another embodiment, the device comprises an actuator capable of providing mechanical stimulation to the solid tumor or to the tissue in the vicinity of the solid tumor when positioned at a proximate location from the tumor on an external skin surface.

Description

[0001] METHOD AND DEVICE FOR DRUG DELIVERY

[0002] Technical field

[0003] The present invention relates, in general terms, to the field of drug delivery. In particular, the specification teaches methods and devices for promoting infiltration of nanoparticles into solid tumors using mechanical stimulation.

[0004] Background

[0005] The growing interest in employing nanotechnology for cancer diagnosis and therapy has accelerated the development of drug delivery system using engineered nanoparticlcs, commonly referred to as nanomedicine. Compared with small molecule and biomacromolecule drugs, nanomedicine has distinctive advantages as they have programmable properties and preferential accumulation at tumor sites via the enhanced permeability and retention (EPR) effect. A major goal of this field is to enhance disease-site targeting and reduced adverse side effects, and thus far, multiple therapeutic nanomedicines including doxorubicin-loaded liposome (Doxil) and paclitaxel-loaded albumin (Abraxane) nanoparticles are used clinically for different cancer indications. Nevertheless, the use of nanomedicine for antitumor purpose remains limited as the nanoparticles need to overcome a number of biological barriers from the intravenous injection site to the action site, which can be classified as nanoparticle-protein interaction, circulation in the blood stream, extravasation from blood vessels and accumulation at tumor site, tumor tissue penetration, cancer cell internalization and intracellular drug release. In particular, the dense extracellular matrix and high interstitial pressure of tumor tissues are known to prevent nanomedicine from reaching its target site at a therapeutically sufficient drug concentration, hence compromising efficacy. Therefore, it is highly desirable to develop nanocarricrs to enhance infiltration efficiency into tumor tissues.

[0006] It is well-known that the physicochemical properties of cancer nanomedicine can affect systemic delivery efficacy and therapeutic outcomes. For example, nanoparticlcs with approximately 100 nm diameter are inherently favorable for improved pharmacokinetics and vascular extravasation, while smaller-sized nanoparticles show greater depth penetration into tumor tissues. Active targeting based on ligand modification can also increase the accumulation of nanoparticles at tumor sites. Nevertheless, this class of strategies typically focuses on one or a few biological barriers, and thus has not led to significant improvement in therapeutic benefits. To tackle this issue, multistage delivery strategy based on size or charge conversion has been proposed to systemically overcome multiple barriers and enable nanoparticles to achieve favorable vascular extravasation and tissue permeability at the same time. Another solution is to manipulate transcytosis and intercellular transport delivery system to enhance the penetration capability of nanomedicine across tumor tissue. However, these aforementioned strategies require complex physiochemical modification which may affect the yield and purity of drugs and may not be fully compliant with Good Manufacturing Practices. This can lead to higher manufacturing costs and drug prices which deter pharmaceutical innovations and clinical use. Another way to boost nanomedicine infiltration into tumor tissues is to employ bioagents such as matrix metalloproteinase to degrade the dense extracellular matrix. However, a major concern of this approach is the likelihood of disrupting intercellular connections and inadvertently increasing the risk of tumor metastasis. The addition of bioagents may also be associated with potential toxicity.

[0007] It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

[0008] Summary

[0009] Disclosed herein is a method of promoting infiltration of a nanoparticle -based anti-cancer drug into a solid tumor of a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anti-cancer drug into the subject.

[0010] The mechanical stimulation may be a compressive force. The compressive force may be a pulsative or periodic compressive force.

[0011] Disclosed herein is a method of inhibiting proliferation of a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of the nanoparticle -based anti-cancer drug into the tumor following administration of the nanoparticle -based anti-cancer drug into the subject.

[0012] Disclosed herein is a method of treating a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anticancer drug into the subject, thereby treating the solid tumor in the subject.

[0013] Disclosed herein is a device for promoting infiltration of a nanoparticle-based anti-cancer drug into a solid tumor of a subject, wherein the device is capable of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticlc-bascd anti-cancer drug into the subject.

[0014] Disclosed herein is a device for mechanical stimulation of a solid tumour in a subject, comprising an actuator operable to apply a compressive force to the tumor or to a tissue in the vicinity of the tumor to mechanically stimulate the tumor.

[0015] Brief description of the drawings

[0016] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0017] Fig. 1 Schematic and characterization of actuator-assisted dynamic mechanical stimulation (AADMS). a, Mechanical squeezing led to higher fluid pressure and an increased amount of dyed fluids leaking out of the tubes, b, Mechanical squeezing led to higher pressure and leaking of dyed fluids into surrounding pork tissues, c, Computational simulation showing an increase in nanoparticle concentration in tumor tissue after DMS. d&e, Schematic diagram (d) and physical image (e) of setup of AADMS. The customed robot consisted of an air pump, pressure regulator, frequency controller and soft pneumatic actuator, which could mechanically stimulate tumor tissue in a pulsed manner, f, Computer aided design (CAD) drawings of soft pneumatic actuator, g. The soft pneumatic actuator could apply mechanical force to the contacted objects through increasing the air pressure inside the cavity, h, Different sizes of 3D-printed soft pneumatic actuator to fit tumors of different sizes, i, Images of pneumatic actuator attached on the tumor tissue of mice, j, Relationship between pressure provided through vacuum pump and correlated to forces applied with AADMS. k, Various modes of AADMS. The total time for AADMS was 5 min and the mode was n / 2 on and n / 2 off. “n” refers to the AADMS cycle including 2s, 4s, 8s and 16s. The maximum force was either 1 N, 2 N, 3 N or 4 N, and the actuation duty cycle remains 50%.

[0018] Fig. 2 AADMS enhanced infiltration of liposome and gold nanoparticles into solid tumors, a, Schematic demonstrating AADMS induced infiltration of liposome nanoparticles (LNPs) into tumor tissues, b, Representative transmission electron microscopy (TEM) image of fluorescence molecule indocyanine green (ICG)-loaded LNPs. Scale bar: 200 nm. c, Representative IVIS imaging of breast tumor mice. LNPs were intravenously injected to the mice at 0 h and subsequently administered by AADMS (5 min, 2s on / 2s off) at the 1.5 h. d, Quantification of the fluorescence signal at the tumor site, showing greater LNP infiltration into tumors with AADMS. e-f, Representative photoacoustic imaging (e) and quantification of the photoacoustic signal (f) at the tumor area (n - 10), indicating -92% increase in photoacoustic signals in AADMS-treated tumors, g, Schematic demonstrating AADMS induced infiltration of gold nanoparticles (GNPs) into tumor tissues. h, Representative TEM image. Scale bar: 50 nm. i, Hydration diameter of GNPs. j, Gold content of tumor tissue at different groups (n = 5) measured by inductively coupled plasma mass spectrometry (ICP-MS). k-1, Representative TEM images (k) and GNPs number density (1) of tumor tissue paraffin section from mice at different groups (n - fO). Data are presented as mean ± standard deviation (SD). Two-sided unpaired Student’s Ltest was used for the calculation of the statistical differences in (d) and (j). Two-sided Welch’s t-test was used for the calculation of the statistical differences in (f) and (1). P value < 0.05 was considered statistically significant, and the number of the label represents the range of R values (*P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001).

[0019] Fig.3 AADMS enhanced infiltration of human serum albumin nanoparticles (HSA NPs) into solid tumors, a, Schematic demonstrating AADMS induced infiltration of HSA NPs into tumor tissues, b, Representative TEM image. Scale bar: 1 pm. c, Diameter distribution of HSA NPs in the dehydrated state, d, Hydration diameter of fluorescence molecule IR780- loaded HSA NPs. e, Representative IVIS imaging of breast tumor mice. The mice were administered with HSA NPs (i.v.) followed by the AADMS. f, Quantification of the fluorescence signal at the tumor site, showing higher HSA NP concentration in AADMS- trcatcd tumors, g&h, 1V1S imaging (g) and quantification of fluorescence signal (h) of tumor tissue (w = 5) collected 12 h post-injection of HSA NPs. i, Fluorescence signal of major organs of mice at different groups (n = 5), indicating no to low significant difference in nanoparticle infiltration to other non-target organs with AADMS. j-k, Representative photoacoustic imaging (J) and quantification of the photoacoustic signal (k) at the tumor area (n — 10). 1, Schematic illustration of AADMS administration on a bilateral tumor model. HSA nanoparticles were intravenously injected into the mouse. AADMS was then applied only to the tumor on the right for 5 mins (maximum force of 2 N with a 2s on / 2s off cycle, m, Representative IVIS imaging of the bilateral tumor mouse at different time points, n, Quantification of the fluorescence signals in the bilateral tumor area (n = 5) showing more significant fluorescence increase in AADMS-treated tumor. Data are shown as mean ± SD. Two-sided unpaired Student’ s r-test was used for the calculation of the statistical differences. There is a significant difference between groups when the P value is lower than 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0020] Fig. 4 Safety evaluation and mode optimization of AADMS. a, Risk evaluation of tumor metastasis induced by different modes of AADMS. The mice were subcutaneously implemented with luciferase-expressed 4T1 cells. When the tumor size was 7-8 mm, the tumor tissue was administered with different modes of AADMS on day 0. Luciferase signal of 4T1 cells was detected using IVIS imaging, b, Luciferase signal ratio on day 14 / day 0 outside of tumor-implanted area, indicating no sign of metastasis after AADMS. c, In vivo biosafety assessment of AADMS at different modes via blood biochemical analysis. Liver function indicators included alanine aminotransferase (ALT), aspartate transaminase (AST), albumin (ALB) and alkaline phosphatase (ALP). Renal function urea nitrogen (BUN) and creatinine (CRE). Ischemia-induced necrosis or cell membrane permeability indicators included Creatine kinase (CK) and lactate dehydrogenase (LDH). The heap map showed similar levels of mice blood biochemical indicators at different modes of AADMS and healthy group, d, Optimization and screening of AADMS modes. The heat map shows the ratio of tumor fluorescence signals at 4 h and 2 h. The 1R780-Ioaded HSA NPs was intravenously injected to the mice at 0 h. After IVIS imaging at the 2 h timepoint, tumor tissues were administered with different modes of AADMS. At the 4 h timepoint, nanoparticle distribution in the mice was determined with IVIS imaging, e, The density of circulating tumor cells (4T1) in the blood of tumor mice with different dynamic stimulation modes. The results show that the optimized stimulation mode did not induce tumor metastasis, f, In vivo confocal images showing the nanoparticlcs leakage from blood vessel into tumor tissue at different mechanical stimulation modes. Mode A acts as control group without any stimulation. Mode B with the continuous stimulation, Mode C with the gently dynamic mechanical stimulation and Mode D with the quick dynamic stimulation on tumor tissue with custom device. Nanoparticles can be more efficient to leakage in mode D.

[0021] Fig. 5 Anticancer therapeutic effect evaluation in vivo, a, Schematic illustrations showing that when the tumor grows to 7-8 mm, it was subject to optimized AADMS (5 min, 2s on / 2s off, maximum force: 2 N) followed by intravenous injection of paclitaxel-loaded HS A NPs (PTX@HSA NPs). The tumor volume and body weight were monitored every three days, b-c, Tumor volume changes at different treatment groups (n = 5), showing therapeutic synergy between nanomcdicinc and AADMS. d, Images of tumors collected at the end of experiments, e, Tumor weight of mice at different groups (n - 5). f, Survival curves of mice at different groups (n = 10). g, Body weight change of 4T1 tumor-bearing mice following treatments with PBS, AADMS, PTX@HSA NPs, or AADMS&PTX@HSA NPs (n = 5). h, H&E staining of tumor sections with different groups, showing a large degree of necrotic and apoptotic cells in tumors treated with nanomedicine and AADMS. Data are presented as mean ± SD. Two-sided unpaired Student’s t-test was used for the calculation of the statistical differences in (c) and (e). P value < 0.05 was considered statistically significant, and the number of the label represents the range of P values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0,0001),

[0022] Detailed description

[0023] The present specification teaches a method of promoting infiltration of an anti-cancer drug into a solid tumor of a subject. The method may comprise the step of providing mechanical stimulation to the subject for a sufficient time and under conditions to promote infiltration of the anti-cancer drug into the tumor following administration of the anti-cancer drug into the subject. The anti-cancer drug may be a nanoparticle-based anti-cancer drug.

[0024] Disclosed herein a method of promoting infiltration of a nanoparticlc-bascd anti-canccr drug into a solid tumor of a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticlc-bascd anti-cancer drug into the subject.

[0025] Without being bound by theory, the inventors have shown that the use of dynamic mechanical stimulation (AADMS) can significantly enhance accumulation and penetration of a diverse type of nanoparticles, including liposomes, gold nanoparticles and albumin nanoparticles with clinically relevant physicochemical properties. The different modes (maximum force: 1 - 4 N, cycle: 2 to 16 s) of AADMS did not increase the risk of tumor metastasis or lead to blood toxicity. Furthermore, the cyclic regimen with 2s on / 2s off and maximum force of 2 N most efficiently promoted the infiltration of paclitaxel-loaded human serum albumin nanoparticles (PTX@HSA NPs) into breast tumor tissues, leading to 36.68% shrinkage in tumor size and an additional eight-day extension in 50% survival period.

[0026] In one embodiment, there is provided a method of promoting infiltration of a nanoparticle- based anti-cancer drug into a solid tumor of a subject, the method comprising the step of a) administering a nanoparticle-based anti-cancer drug into a subject; and b) providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor in the subject.

[0027] The term "nanoparticle-based anti-cancer drug" may refer to a small particle of less than about lOOOnm that is associated with an anti-cancer drug. The small particle may be a liposome, extracellular vesicle (such as an exosome), virosome, virus-like particle (VLP), plasma membrane vesicles (PMVs), niosomes, lipid core peptides (LCPs), immuno stimulating complexes (ISCOMs), polymer based nanoparticles (e.g. poly(D,L-lactic-co- glycolic acidj(PLGA) nanoparticles), metal nanoparticles (e.g. gold nanoparticles) or protein nanoparticles (e.g. albumin nanoparticles). The anti-cancer drug may be attached to the nanoparticlcs via covalent bonds or non-covalcnt bonds or may be contained within the nanoparticles. The small particle may be about 10 nm to about 100 nm, about 100 nm to 200 nm, about 200 nm to about 300 nm, about 300 to about 400 nm, about 400 nm to about 500 nm, about 500 nm to about 600 nm or about 600 nm to about 700 nm. In one embodiment, the small particle is about 50 nm to about 500 nm. In one embodiment, the small particle is about 50 nm to about 400 nm. In one embodiment, the small particle is about 50 nm to about 300 nm. In one embodiment, the small particle is about 40 nm to about 200 nm. In one embodiment, the small particle is about 40 nm to 100 nm. In one embodiment, the small particle is about 40 nm to 50nm. In one embodiment, the small particle is about 100 nm to 200 nm. The small particle may, for example, be a liposome nanoparticle (around 40 nm), gold nanoparticle (around 50 nm), or albumin nanoparticle (around 200 nm).

[0028] The term “tumor,” as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. By “non-metastatic” is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a Stage 0, 1, or 11 cancer, and occasionally a Stage 111 cancer. By “early stage cancer” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or II cancer. The term “late stage cancer” generally refers to a Stage III or Stage IV cancer, but can also refer to a Stage II cancer or a substage of a Stage II cancer. One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer. In an embodiment, the cancer is a breast cancer, prostate cancer, head and neck cancer, oral cancer, sarcoma or a cancer of the lymph node. In an embodiment, the cancer is breast cancer or a sarcoma.

[0029] The modes of mechanical stimulation / actuation may include ultrasound waves, sound waves, piezoelectric, resistive, thermal expansion and contraction, fluidic, pneumatic, actuation of shape memory alloys or controlled phase transition. The mechanical stimulation may be generated by electrohydraulic, piezoelectric and / or electromagnetic techniques, or pneumatically.

[0030] The mechanical stimulation may be provided prior to, concurrently or following administration of a drug to the subject.

[0031] The method may comprise providing mechanical stimulation to the subject in the vicinity of the solid tumor. The method may comprise providing mechanical stimulation to the subject in the vicinity of the tumor or tissue surrounding the tumor. The mechanical stimulation that is provided to the tumor may increase vascular pressure in the vicinity of the tumor or within the tumor as compared to a reference (e.g. the vascular pressure in the vicinity of the tumor or within the tumor when no mechanical stimulation is applied). The increase in vascular pressure may be transient. This may promote infiltration of the nanoparticlc-bascd anticancer drug into the tumor. This may also promote uptake of the nanoparticle-based anticancer drug by the tumor cells.

[0032] The method may comprise providing mechanical stimulation to the subject at one or more proximate locations from the tumor (i.e. one or more sites in the vicinity of the tumor) on an external skin surface of the subject. The method may comprise contacting the subject with one or more devices capable of providing mechanical stimulation at one or more proximate locations from the tumor on an external skin surface of the subject. The mechanical stimulation may be applied, for example, at 1, 2, 3, 4, 5, 6 or more proximate locations.

[0033] The mechanical stimulation may be applied directly to the tumor, or to the tissue surrounding to the tumor, or indirectly to tissue in the vicinity of the tumor. The mechanical stimulation may comprise compressive pressure applied directly to the tumor or to the tissue surrounding to the tumor, or indirectly to tissue in the vicinity of the tumor.

[0034] Tn one embodiment, the mechanical stimulation is a compressive force applied to the tumor or to the tissue (e.g. skin tissue) surrounding the tumor.

[0035] The method may comprise providing a force of about IN to about ION, or more, to the solid tumor. The method may, for example, comprise providing a force of about IN to about 9N, about IN to about 8N, about IN to about 7N, about IN to about 6N, about IN to about 5N, about IN to about 4N, about IN to about 3N or about IN to about 2N to the solid tumor. The method may comprise providing a pressure of about IN, about 2N, about 3N, about 4N, about 5N, about 6N, about 7N, about 8N, about 9N, about ION, or more, to the solid tumor.

[0036] The method may comprise providing the force periodically to the solid tumor. For example, the method may comprise providing the pressure for a period of Is, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, I ls, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, or more, followed by a rest phase. The rest phase may, for example, be Is, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, Ils, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, or more.

[0037] The method may comprises the step of administering the nanoparticle-based anti-cancer drug to the subject.

[0038] The term “administering” as used herein refers to contacting, applying, delivering, providing or feeding an effective amount of the nanoparticlc-bascd anti-cancer drug to a subject in need by any appropriate means. Modes of administration may be parenteral, enteral and topical. Parenteral administration is usually by injection, and includes, without limitation, intravenous, intramuscular", intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the nanoparticle-based anti-cancer drug is administered intravenously to the subject.

[0039] The term “chemotherapy” refers to a therapy of a human or animal with one or more chemotherapeutic agents, which inhibit or abrogate cell growth and cell division, namely, the therapy is taken as a cell proliferation inhibitor or is used for inducing cell death (cell apoptosis). Compared with normal cells, cancer cells grow and divide uncontrollably so that the chemotherapy should be more effective to the cancer cells.

[0040] The anti-cancer drug may include chemotherapeutic agents, representative examples of which include antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (c.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimctabolitcs, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.

[0041] Specific examples of chemotherapeutic agents include erlotinib (TARCEVA, Genentech / OSI Pharm.), bortezomib (VELCADE, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX, AstraZeneca), sunitib (SUTENT, Pfizer / Sugen), letrozole (FEMARA, Novartis), imatinib mesylate (GLEEVEC, Novartis), finasunate (VAT ALANIB, Novartis), oxaliplatin (ELOXATIN, Sanofi), 5-FU (5 -fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE, Wyeth), Lapatinib (TYKERB, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR, Bayer Labs), gefitinib (TRESS A, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamidc; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5a- reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostatdolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mclphalan, novcmbichin, phcncstcrinc, prcdnimustinc, trofosfamidc, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin y1 T and calicheamicin col l (Angew Chem. Inti. Ed. Engl. 1994 33:183-186); dyncmicin, including dyncmicin A; bisphosphonates, such as elodronate; an cspcramicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, dctorubicin, 6-diazo-5-oxo-L-norlcucinc, ADR1AMYC1N (doxorubicin), morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), Doxil (doxorubicin HC1 liposome injection), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin. zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercap topurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxy uridine, doxifluridine. enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mcpitiostanc, tcstolactonc; anti-adrenals such as aminoglutcthimidc, mitotanc, trilostanc; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; cniluracil; amsacrinc; bcstrabucil; bisantrcnc; edatraxate; dcfofaminc; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, TIL), and TAXOTERE (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR (gemcitabine); 6-thioguaninc; mcrcaptopurinc; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA); ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromcthylornithinc (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above. Chemotherapeutic agents also include (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifme citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide. nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxy me sterone, all transrctionic acid, fenretinide, as well as troxacitabinc (a 1,3-dioxolanc nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-a, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0042] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / B iogen Idee), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, cfalizumab, cpratuzumab, erlizumab, fclvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pcxclizumab, ralivizumab, ranibizumab, rcslivizumab, rcslizumab, rcsyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab and visilizumab.

[0043] Disclosed herein is a method of inhibiting proliferation of a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of the nanoparticle -based anti-cancer drug into the tumor following intravenous injection of the nanoparticle -based anti-cancer drug into the subject.

[0044] Methods of treating a solid tumor are also provided herein.

[0045] Disclosed herein is a method of treating a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anticancer drug into the subject, thereby treating the solid tumor in the subject.

[0046] In one embodiment, there is provided a nanoparticlc-bascd anti-cancer drug for use in treating a solid tumor in a subject, wherein mechanical stimulation is to be administered to the subject in the vicinity of the solid tumor following administration of the nanoparticlebased anti-cancer drug so as to promote infiltration of the nanoparticle-based anti-cancer drug into the tumor.

[0047] In one embodiment, there is provided the use of a nanoparticle-based anti-cancer drug in the manufacture of a medicament for treating a solid tumor in a subject, wherein mechanical stimulation is to be administered to the subject in the vicinity of the solid tumor following administration of the medicament so as to promote infiltration of the nanoparticle-based anticancer drug into the tumor.

[0048] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or condition and / or adverse effect attributable to the disease or condition. These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include: (a) inhibiting the disease or condition, i.e., arresting its development; or (b) relieving the disease or condition, i.e., causing regression of the disease or condition.

[0049] By “effective amount,” in the context of treating or preventing a disease or condition is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for the treatment or prevention of that disease or condition. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0050] Disclosed herein is a method of treating a solid tumor in a subject, the method comprising the step of a) administering a nanoparticle -based anti-cancer drug to a subject; and b) providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of the nanoparticle-based anticancer drug into the tumor, thereby treating the solid tumor in the subject.

[0051] Disclsoed herein is a device for promoting infiltration of a nanoparticle-based anti-cancer drug into a solid tumor of a subject, wherein the device is capable of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anti-cancer drug into the subject.

[0052] The device may be capable of pneumatically or hydraullically generating mechanical stimulation. The device may comprise a control system comprising at least a pump and a control circuit electrically connected to the pump.

[0053] The device may further comprise an actuator that is connected to the pump. The actuator may be capable of providing mechanical stimulation to the solid tumor or to the tissue in the vicinity of the solid tumor when positioned at a proximate location from the tumor on a external skin surface. The device or the actuator may be 3D printed and may be flexible so as to conform to the surface of the solid tumor or to the tissue around the tumor. The actuator of the device can be sized to cover part or all of the tumor to mechanically stimulate part or all of the tumor. In an embodiment, the device is a device configured to surround a tumor (e.g. a breast tumor) of the subject when applied to the subject.

[0054] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0055] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0056] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0057] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0058] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] Certain embodiments of the invention will now be described with reference to the following examples which arc intended for the purpose of illustration only and arc not intended to limit the scope of the generality hereinbefore described.

[0061] EXAMPLES

[0062] Example 1: Design and fabrication of mechanical actuator

[0063] Design and printing of soft pneumatic actuator

[0064] The TPU filament was purchased from Recreus. CAD software (Solidworks 2020, Dassault Systemes) was used to design the dynamic mechanical actuator. The CAD part file was run through a three-dimensional (3D) slicing software (Simplify3D LLC, OH) and the generated G-code file was loaded into a 3D printer (Creator pro, Flashforge) for the fabrication of soft pneumatic actuator. The soft pneumatic actuator was printed using the TPU filament with a shore hardness of 60 A. The filament was extruded using Diabase Engineering Flexion Extruder with a 0.5 mm nozzle diameter and 1.37 extrusion multiplier, at 200°C printing temperature and 20 mm / s printing speed. The layer height was set to 0.2 mm to guarantee printing precision.

[0065] Design and fabrication of mechanical-driven robot Pneumatic pressure is generated from a pump (550W-8L, OUTSTANDING). The pressure value can be regulated manually through a regulator (1R1000-01BG, S1MA1TE). To periodically activate the 3D printed soft actuator, a solenoid valve (P12N2N651A, CZEVRCH) is introduced to switch the pneumatic pressure. The circuit is built to control the solenoid valve. An Arduino controller is used to control the cycle of actuation.

[0066] Computer simulation of theoretical model of nanoparticle transport in a tumor

[0067] To demonstrate the mechanism of AADMS, the computer simulation was carried out as followed:

[0068] 1. Mathematical model

[0069] The solid tumor can be treated as a porous medium since the inter-capillary distance is typically 2~3 orders lower than the length scale of drug transport. The incompressible, Newtonian interstitial fluid flow is governed by the mass and momentum conservation equations, as where p and p are the density and viscosity of the interstitial fluid, respectively, p is the interstitial fluid pressure, and v is the velocity, K refers to tissue permeability. T is the stress tensor. The fluid loss from the blood to tissue (Fb) is described by Starling’s law, as in which Kbvis the hydraulic conductivity of the microvasculature wall. Sbvstands for the surface area of the microvasculature wall per tissue volume. pbis the vascular pressure. crbis the average osmotic reflection coefficient for proteins in plasma. are the osmotic pressure of the blood and interstitial fluid, respectively. The drug concentration (Ct) in tumor is determined by the diffusion and convective transport with the interstitial fluid flow, drug exchange between the blood and tumor, and release dynamics, as where DNPis the diffusivity of nanoparticles in the tumor. PNPis the transvascular permeability of nanoparticles. crNPis the osmotic reflection coefficient of nanoparticles. / crelis the drug release rate. Pe is the Peclet number, defined as is nanoparticle concentration in the blood, governed by where kc\cstands for the plasma clearance of nanoparticles.

[0070] 2. Model parameters

[0071] Given the delivery time window is much smaller as compared to the tumor growth rate, the properties of the tumor and nanoparticles are assumed to be constant. The plasma clearance of nanoparticles (kclc) is set to be 8.02 x 10-6s-1, since the half-life in plasma was measured as 24-hr . The diffusivity (DNP) of 100nrn nanoparticle is 9.0 x 10’12m2 / s. It is assumed that the squeezing force is translated to vascular pressure (pb). Therefore, this pressure can be calculated by where pb 0is the original vascular pressure. pb msis the pressure induced by mechanical squeeze, determined by the squeezing force.

[0072] 3. Model geometry The mathematical modeling of nanoparticle delivery is performed in an idealized tumor geometry with a radius of 0.5 mm . The final computational mesh consists of 1.5 x 104triangular elements, which are obtained after the mesh independence test.

[0073] 4. Boundary conditions

[0074] Since this is an isolated tumor used to mimic the real physical experiment, the gauge pressure and nanoparticle flux on the tumor surface are taken to be 0.

[0075] 5. Numerical method

[0076] The governing equations of interstitial fluid flow and drug transport are implemented in the finite element method-based numerical modeling code package, COMSOL, for generating the numerical solutions. Given that 10 ug of drugs were injected into mice whose average blood volume was 2 mb, the initial drug concentration in blood is set to be 5.0 μg / mL. The concentration in tumor is 0 at the beginning of the treatment.

[0077] Example 2; Mechanical stimulation of tissue to promote nanoparticle delivery

[0078] Materials

[0079] Chloroauric acid trihydrate , ascorbic acid, sodium citrate dihydrate and glutaraldehyde were purchased from Sigma- Aldrich (Germany). Distearoyl phosphatidylcholine (DSPC), cholesterol, human serum albumin (HSA) powder and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000] (DEPE-PEG2000) were purchased from Merck (Germany). Chloroform and dimethyl sulfoxide (DMSO) were supplied by TCI (Japan). Indocyanine green (ICG) and 2-[2-[2- chloro-3-[(1 ,3-dihydro-3,3-dimethyl-1 -propyl-2h-indol-2-ylidene)ethylidene]-1 - cyclohexen-l-yl]ethenyl]-3,3-dimethyl-l-propylindolium iodide (IR780) were obtained from Ruixibio (China).

[0080] Preparation of liposome nanoparticles Liposome nanoparticles were prepared according to previously reported method with minor modification. In detail, 2.4 mg DSPC, 1.8 mg DEPE-PEG2000 and 0.7 mg cholesterol were dissolved into 2.5 mL of chloroform in a round bottom flask. Chloroform was removed through roto evaporation and formation of a thin film can be observed on the inside wall of round bottom flask. Afterwards, the aqueous solution containing fluorescence molecules ICG was added to the round bottom flask. The mixture solution was placed on the ultrasonic crusher (100 w, Is on / ls off) for 5 min. To obtain uniform nanoparticles, the liposome solution was filtered using liposome extruder 20 times with a 200 pm filter membrane.

[0081] Preparation of gold nanoparticles

[0082] Gold nanoparticles were fabricated based on reported method. 15 nm of gold nanoparticles was first synthesized with a citrate reduction method. Briefly, (Chloroauric acid trihydrate) was added into 100 mL of 18.2 MΩ cm Milli-Q water, which remained boiling for 3 min. Then, 3 mL of 1% sodium citrate was quickly (within 2s) injected while stirring vigorously (200 rpm / min). After the color completely turned from x into cherry red, the solution continued to boil for 0.5 h. After the addition of 5 mL of 0.1% ascorbic acid, the stirring mixture was cooled to room temperature. The gold nanoparticles were washed through centrifugation (10 min, 7500 g) and maintained at a final volume of 100 mL which was used as seed solution. 50 nm of gold nanoparticles were prepared by the seeded growth method. Briefly, 2.5 mL of was diluted with 8 mL of seed solution and 39.5 mL of Milli-Q water. Then the reductant solution (45 mL of 0.1% ascorbic acid and 5 mL of 1% sodium citrate) was slowly dropped into the mixture in 45 min. The obtained gold nanoparticles were centrifuged (10 min, 5700 g) to remove any residual reductant reagents.

[0083] Preparation of HSA nanoparticles

[0084] HSA nanoparticles were fabricated according to previously reported method. In detail, 15 mg HSA powder was first dissolved in 150 uL deionized water. 1R780 was dissolved in DMSO with a final concentration of 2.5 mg / mL. Next, 25 uL of IR780 solution was slowly added to the HSA solution with a fully stirring. Afterwards, 100 uL glutaraldehyde solution (0.5%) was dropwise to above solution and the mixture was stirred for 2 h. Finally, the nanoparticles were washed using an ultrafiltration tube (pore size: 10 kDa) for three times and stored at 4°C.

[0085] Animal studies

[0086] Healthy BALB / c mice (female, 6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, Subcutaneous breast cancer model was obtained by injecting 1 x 1064T1 cells into the right flank site. The tumor volume was calculated according to the formula “lengthxwidth2 / 2”. The body weight and tumor volume were recorded every three days. When tumor diameter reached 6-7 mm, different species of nanoparticles were intravenously injected to mice through the tail vein. Afterwards, the tumor tissue was subjected to actuator-assisted dynamic mechanical stimulation (AADMS) for 5 minutes with different force (1 N, 2 N, 3 N and 4 N) and cycle (2s, 4s, 8s and 16s). The nanoparticles infiltration at different time points in the tumor site was observed using in vivo fluorescence imaging system (1V1S Spectrum, Perkin Elmer) and multispcctral optoacoustic tomography (MOST invision 128, iThera). All the animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of National Center for Nanoscience and Technology and approved by the Institutional Animal Care and Use Committee (IACUC).

[0087] Statistical analysis

[0088] Statistical analysis was generated with GraphPad Prism (version 8.0, GraphPad). Data with the equal variance was analyzed by two-sided unpaired Student’s t-test. For data with significantly different variances, two-sided Welch’s t-test was used for comparison between groups. P values less than 0.05 were considered statistically significant and noted in each figure or their legend. P value ranges were exhibited with following manner: All samples were blindly randomized, and no sample was excluded from analysis. Data are presented as mean ± standard deviation (SD) and all individual data points are also shown.

[0089] Results

[0090] Robot design and configuration As illustrated in Fig. la, with permeable tubes, higher pressure due to mechanical squeezing led to an increased amount of dyed fluid leaking into the surrounding environment. To further demonstrate this in a tissue setting, permeable tubes were inserted into pork tissue. Fig. lb showed that with mechanical squeezing, there are dyed fluids leaking into surrounding tissues and the greater the magnitude of squeezing, the stronger the fluid flow pressure, causing greater dye color intensities. Inspired by this phenomenon, using a drug delivery mathematical model, the delivery outcomes of nanoparticles with and without mechanical squeezing regimen consisting of a 2 s of pulsatile mechanical squeeze followed by a 2 s rest period for a total of 5 minutes were first compared. The maximum squeezing force was 2 N which was translated to a higher transient vascular pressure. The result in Fig. 1c shows the time course of nanoparticle concentrations in the tumor with and without actuator- assisted dynamic mechanical stimulation (AADMS). AADMS effectively enhanced the infiltration of nanoparticles from the leaky vasculature into tumor tissue, leading to a concentration increase in tissues while eventually declining two hours after dynamic mechanical stimulation (DMS) ended and supporting the use of DMS to improve drug delivery to tumors.

[0091] To achieve pre-programmed mechanical actuation of tumor tissues, AADMS was proposed as a method to enhance nanoparticle infiltration into solid tumor tissues (Fig. Id). The specialized robot system consisted of an air pump, pressure regulator, frequency controller and 3D-printed soft pneumatic actuator which applied pulsatile mechanical forces to the tumor tissue (Fig. le). Increasing the air pressure inside the soft actuator provided sufficient force to contract objects (Fig. lf-g). The 3D-printed soft pneumatic actuator could be size- customized for precise fitting onto tumors (Fig Ih-i). Importantly, the magnitude of mechanical force applied by the robotic system could be regulated by adjusting air pressure inside soft actuator (Fig. 1j). The robotic actuator applied a periodic and dynamic mechanical force onto tumor tissue with a maximum mechanical force ranging between 1-4 N. By switching the controller, the AADMS frequency can be controlled from 1 / 16 Hz to 1 / 2 Hz, and the actuation duty cycle remains 50% (Fig. Ik).

[0092] AADMS enhances infiltration of liposome and gold nanoparticles into solid tumor Liposome-based nanocarriers are widely used to deliver anti-cancer chemotherapy. In particular, Doxil is an FDA-approved doxorubicin-loadcd liposome nanoparticlcs that can significantly decrease the adverse toxicity of chemotherapeutic drugs. Motivated by this clinical use of liposomes, it was first evaluated whether AADMS could enhance the infiltration of liposome nanoparticles (LNPs). LNPs were intravenously injected into the tail vein of the breast tumor-bearing mice (Fig. 2a) followed by cyclic mechanical squeeze (2s on / 2s off) on the tumor tissue for 5 min. To track the localization of LNPs in vivo, indocyanine green (ICG) which functioned both as a fluorescence and photoacoustic agent was encapsulated into LNPs. The ICG-loaded LNPs possessed uniform morphology with a diameter in the dehydrated state of 40 nm and a hydrodynamic diameter of 96 nm (Fig. 2b). When ICG-LNPs were intravenously injected to the mice, only weak fluorescence signals were observed at the tumor site (Fig. 2c-d), indicating that only a small quantity of nanoparticlcs accumulated at the tumor tissue. However, with AADMS, there was a 2.2-fold increase in fluorescence signal at the tumor site 8 h post-injection, supporting that AADMS could greatly enhance the accumulation of LNPs in solid tumor tissue. Photoacoustic imaging was also used to assess the penetration of LNPs in the solid tumor. As shown in Fig. 2e, LNPs penetrated more deeply into tumor tissue with AADMS. The photoacoustic signal of tumor tissue in AADMS -heated group was 91.98% higher than that in untreated group (Fig. 2f).

[0093] Gold nanoparticles (GNPs) are widely used to anti-tumor therapeutic attributing to their inert photothermal performance and modifiable surfaces. In a clinical trial of gold nanoshell- localized photothermal ablation, 13 of the first 15 cancer patients who received treatment did not detect any signs of cancer after one year of treatment. Motivated by this, the infiltration of GNPs into the tumor tissue was evaluated with AADMS (Fig. 2g). The GNPs had a diameter in the dehydrated state of 50 nm and a hydrodynamic diameter of 54 nm. (Fig- 2h-i). After intravenous injection of GNPs into tail vein, the tumor tissue was subject to periodic mechanical stimulation with a 2s on / 2s off mode for 5 mins. 24 h post-AADMS treatment, the tumor tissues were collected and analyzed quantitatively for gold content with inductive coupled plasma-mass spectrometer (ICP-MS) which found that AADMS improved GNPs infiltration into tumor tissues by 31.76% (Fig. 2j). Electron microscopic images also revealed that with AADMS, there was 167% more GNPs in tumor slices (Fig. 2k and 1). AADMS enhances infiltration of albumin nanoparticles into solid tumor

[0094] To test the universality of AADMS to improve infiltration of nanoparticle with different material composition into tumor tissues, human serum albumin nanoparticles (HSA NPs) were next injected intravenously through the tail vein before applying AADMS (Fig. 3a). Albumin nanoparticles was chosen as drug carrier because of its safety and biocompatibility. In particular, paclitaxel-loaded albumin (Abraxane) nanomedicine have been approved by FDA and can substantially decrease the adverse toxicity of chemodrug in clinical trials. To monitor in vivo distribution of HSA nanoparticles, IR780 which functioned both as a fluorescence and photoacoustic agent was loaded. The IR780-loaded HSA NPs had a diameter in the dehydrated state of 189 nm (Fig. 3b-c) and a hydrodynamic diameter of 193 nm (Fig. 3d). The results in Fig. 3e and f showed that HSA NPs were 38% more likely to infiltrate and accumulate at the tumor tissue 12-h post AADMS (2s on / 2s off mode for 5 mins). Fluorescence analyses of ex vivo tumor tissues similarly found that AADMS-treated tissues had 73% more HSA NPs at the 12 h time point (Fig. 3g-h). Importantly, AADMS did not substantially change the distribution of HSA NPs in major organs (Fig. 3i). Photoacoustic imaging showed that AADMS-treated tumor tissues had 79% higher photoacoustic signal intensity (Fig. 3i) than that in the unstimulated control group (Fig. 3j). To further validate this, a bilateral tumor model was established where AADMS was only administered to the tumor on the right (Fig. 31). Fig. 3m-n showed a noticeable rise in fluorescence signal only in the tumor on the right, indicating that AADMS had a significant impact in promoting nanoparticle infiltration. Collectively, the data with the three nanoparticle species suggests that AADMS could function as a material-agnostics method using mechanical squeezing to improve the infiltration and accumulation of nanoparticles in tumor tissues.

[0095] AADMS mode optimization for albumin-based nanoparticles

[0096] Tumor metastasis can occur due to destruction of intercellular connections. Mechanical disruption to the extracellular matrix spaces surrounding tumor spaces is a potential safety consideration for AADMS. The risk of tumor metastasis associated with AADMS was evaluated through detecting luciferase signal distribution of 4T1-Luc cells in the mice. As shown in Fig. 4a, when the tumor tissues were administered with AADMS at different modes, the luminescent signals cells were observed only at tumor area 7 and 14 days post- administration of AADMS. The ratio of signal outside of the tumor site on day 14 (after AADMS treatment) and day 0 (before AADMS treatment) is close to 1, indicating that there was no statistically significant increase in luminescent signals elsewhere in the animals except at the tumors and an extremely low risk of tumor metastasis induced by AADMS (Fig. 4b). Next, the biosafety of AADMS at different modes was assessed with blood biochemical analysis. As shown in Fig. 4c, the levels of the blood biochemical indicators (liver and renal functions, myocardial proteins related to myocardial ischemia-induced necrosis or cell membrane permeability) in healthy and AADMS -treated mice were similar i.e., ratio closes to 1. Using values of pressure during mammogram, a gold-standard method for breast imaging which applies significant pressure on the breast tissues, it is estimated that AADMS applies a pressure of -0.6 kPa in the optimized mode. This value was ~23x less than that during clinical mammogram (averaging 14 kPa). The pressure during AADMS is likely to not cause pain or induce discomfort according to a report that a pressure of 10.3- 13.5 kPa is accepted with respect to reported pain during mammogram. Finally, the force and cycle of AADMS were optimized based on an increase in fluorescence signal of HSA NPs in tumor area. As shown in Fig. 4d, when the tumor tissue was stimulated by the 2N of force in the 2s on / 2s off mode, the infiltration of nanoparticles in tumors can be maximized. Therefore, this optimized condition was used for subsequent anti-tumor treatment using HSA NPs in Fig. 5.

[0097] In vivo assessment of anti-tumor therapeutic effect

[0098] Encouraged by the significant improvement of nanoparticle infiltration into tumor tissues with AADMS, the anti-tumor effect of AADMS was next evaluated on breast tumor model. As illustrated in Fig. 5a, when tumor size reaches about 7-8 mm, paclitaxel-loaded HSA NPs (PTX@HSA NPs) were intravenously injected to the mice, followed by the AADMS on tumor tissue for 5 min at the optimized mode (2s on / 2s off, maximum force: 2 N). The tumor size and body weight were monitored every three days. As shown in Fig. 5b-c, the tumor growth rates in the control and AADMS group were comparable, indicating that AADMS could not substantially inhibit the tumor growth by itself, as expected. Relatively to the control group on day 24, there was a 40.1 % reduction in the tumor sizes of mice treated with PTX@HSA NPs while therapeutic synergy between PTX@HSA NPs and AADMS resulted in a 62.1% reduction in tumor size compared to control (Fig. 5c). The tumor weight on day 24 in AADMS&PTX@HSA NPs group was 37.1% lighter than that in PTX@HSA NPs group, further confirming the synergistic effect of AADMS nanomedicine (Fig. 5d-e). At day 39, 50% of the mice treated with PTX@HSA NPs survived while the addition of AADMS extended this statistic to day 47 (Fig. 5f). it was also found that AADMS alone or in combination with PTX@HSA NPs did not lead to sudden change in body weight (Fig. 5g), or morphology difference of major organs, indicating excellent biosafety. H&E staining of tumor tissue at the end of treatment indicated that the majority of the tumor cells of mice treated with combinatorial AADMS and PTX@HSA NPs were severely apoptotic and necrotic, while PTX@HS A NP treated group exhibited partially damaged tumor tissue (Fig. 5h). Taken together, all the above results supported enhanced therapeutic efficacy of nanomedicine with AADMS.

[0099] DISCUSSION

[0100] Nanocarrier is an active area of research in the past three decades for cancer therapy. Nanomcdicinc is promising as it has numerous advantages including reduced toxicity, prolonged circulation and controllable release. Nevertheless, the unique structure of tumor tissues including dense extracellular matrix and high interstitial fluid pressure limits efficient infiltration and accumulation of nanomedicine. To tackle this issue, tremendous investments and progress have been made to optimize physicochemical properties (size, surface charge, stiffness, shape, etc.), multistage delivery and transcytosis-intercellular transport strategy. Although these strategies have increased the infiltration ability of nanomedicine in tumor tissue, chemical modifications can be costly, complex, and non-compliant with Good Manufacturing Practices which affects clinical translation.

[0101] In this study, actuator-assisted dynamic mechanical stimulation (AADMS) is found to be a novel approach to enhance infiltration of nanoparticles with clinically relevant physiochemical properties into solid tumors. Based on computer simulation results, it was first demonstrated that AADMS could enlarge vascular gaps through increasing vascular pressure in the leaky tumor-associated vasculature, thus driving infiltration of various types of nanoparticles (liposome, gold, and human serum albumin) into tumor tissues. This suggests that the strategy of mechano-therapy is not dependent on the nanoparticles species and might be further optimized as a universal method to improve nanomcdicinc delivery efficiency. Although degrading the extracellular matrix may increase the infiltration and concentration of nanomcdicinc in tumor tissue, this approach can result in tumor metastasis due to destruction of intercellular connections. It was found that AADMS at different modes did not lead to tumor metastasis and evident fluctuations in blood biochemical parameters. Moreover, AADMS at optimized mode did not cause sudden change of body weight or obvious damage to major organs. This also further supports AADMS as a safe method to enhance nanoparticle infiltration into solid tumors. More importantly, the therapeutic synergy between nanomedicine and AADMS was found to significantly improve the antitumor effect of PTX@HSA NPs and even extended survival period of animals.

[0102] This is the first instance that the concept of DMS or mechanotherapy has been applied to enhance the infiltration of nanoparticles into solid tumor tissues. In contrast with traditional strategics, this method bypasses complex physicochemical modifications and avoids the risk of tumor metastasis. Relative to other methods that combine nanomedicine and physical stimulations such as magnetic hyperthermia and photothcrmal therapy, AADMS docs not require bulky equipment infrastructure like an electromagnet and laser. For instance, AADMS can be powered using batteries and may even be administered either during chemodrug infusion in the clinics or as an at-home kit based on the findings that AADMS could enlarge the vasculature pores by a few hours after stimulation. As a device for external device, AADMS also faces lower regulatory barriers for clinical translation unlike a new nanomedicine compound.

[0103] One application is to convert an inoperable breast cancer to an operable one by combining neoadjuvant chemotherapy with AADMS and enable the patient to undergo curative resection. The other application is to make use of enhanced nanomedicine infiltration into breast tissues to shrink tumors and convert a non-conservable breast cancer resection to one where a patient can conserve her breast. Beyond breast cancer, AADMS could be useful for other external tumors such as sarcoma. Sarcomas arc challenging cancers with a predilection for pediatric and young adult populations and account for >20% of tumors in the young. They typically show poor outcomes, partly attributing to poor penetration of drugs into these mesenchymal tumors. Doxorubicin-based treatment is the standard of care in most sarcomas, while paclitaxel can be used in a specific and highly aggressive sarcoma subtype, the angiosarcomas. As sarcomas can also commonly be found in the limbs and extremities, AADMS can be applied to such tumors in combination with intravenous nanomedicine- based chemotherapy with the aim of converting inoperable tumors to operable, or converting a non-salvagcablc limb to a salvageable one, thereby avoiding amputation, with the aim of cure. In situations where there is metastatic disease and the aim of treatment may not be curative, there is often significant pain or morbidity from these locations. Once again, AADMS can allow better penetration of nanomedicine into these tumors to enhance local disease control and symptom control, thus improving quality of life.

[0104] Attributing to the need for physical contact between the robotic device and tumor tissues for AADMS, this technique has only been tested on external breast tumor tissues. However, it can also be applied to internal, deep-tissue tumor through an implant or material that can be wireless actuated such as magnetic hydrogel.

[0105] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of promoting infiltration of a nanoparticle-based anti-cancer drug into a solid tumor of a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anti-cancer drug into the subject.

2. The method of claim 1, wherein the mechanical stimulation is a compressive force applied to the tumor or to a tissue surrounding the tumor, or to a tissue in the vicinity of the tumor.

3. The method of 2, wherein the mechanical stimulation is a compressive force applied to the tumor or to a tissue surrounding the tumor.

4. The method of any one of claims 1 to 3, wherein the mechanical stimulation is capable of increasing vascular pressure within the tumor or in the vicinity of the tumor as compared to a reference.

5. The method of any one of claims 1 to 4, wherein the method comprises providing mechanical stimulation to the subject at one or more proximate locations from the tumor on an external skin surface of the subject.

6. The method of any one of claims 1 to 4, wherein the method comprises providing mechanical stimulation at one or more locations internal to the subject in the vicinity of the tumor.

7. The method of any one of claims 2 to 6, wherein the force is about IN to about 4N.

8. The method of claim 7, wherein the force is a constant force.

9. The method of claim 7, wherein the force is a pulsative or periodic force.

10. The method of claim 8 or 9, wherein the force is applied for a duration of about 1 sec to about 8 sec in each period.

11. The method of claim 9 or 10, wherein the force is applied for a duration of about 1 sec to about 8 see in each period followed by a rest phase of about 1 sec to about 8 sec.

12. The method of any one of claims 1 to 11 , wherein the mechanical stimulation is provided for up to 2 h following administration of the nanoparticle.

13. The method of any one of claims 1 to 12, wherein the solid tumor is a sarcoma, a lymphoma or a tumor of the breast, prostate, mouth or head and neck.

14. The method of any one of claims 1 to 13, wherein the method comprises the step of administering the nanoparticle-based anti-cancer drug to the subject.

15. The method of claim 14, wherein the method comprises administering the nanoparticle - based anti-cancer drug intravenously to the subject.

16. The method of any one of claims 1 to 15, wherein the nanoparticlc-bascd anti-cancer drug is about 40-200 nm in size.

17. A method of inhibiting proliferation of a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of the nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticle-based anti-cancer drug into the subject.

18. A method of treating a solid tumor in a subject, the method comprising the step of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anticancer drug into the tumor following administration of the nanoparticle-based anticancer drug into the subject, thereby treating the solid tumor in the subject.

19. A device for promoting infiltration of a nanoparticle-based anti-cancer drug into a solid tumor of a subject, wherein the device is capable of providing mechanical stimulation to the subject in the vicinity of the solid tumor for a sufficient time and under conditions to promote infiltration of a nanoparticle-based anti-cancer drug into the tumor following administration of the nanoparticie-based anti-cancer drug into the subject.

20. The device of claim 19, wherein the device is capable of pneumatically or hyduallically generating mechanical stimulation.

21. The device of claim 19 or 20, wherein the device comprises an actuator capable of providing mechanical stimulation to the solid tumor or to the tissue in the vicinity of the solid tumor when positioned at a proximate location from the tumor on a external skin surface.

22. A device for mechanical stimulation of a solid tumour in a subject, comprising an actuator operable to apply a compressive force to the tumor or to a tissue in the vicinity of the tumor to mechanically stimulate the tumor.

23. The device of claim 22, wherein the actuator is configured to apply a compressive force of about IN to about 4N to the tumor or to the tissue in the vicinity of the tumor.

24. The device of claim 22 or 23, wherein the actuator is operable pneumatically or hydraulically.

25. The device of any one of claims 22 to 24, wherein the actuator is configured to apply a compressive force when positioned at a proximate location from the tumor on a external skin surface of the subject.

26. The device of any one of claims 22 to 25, comprising a frequency controller connectable to the actuator and operable to determine a frequency of the applied force.

27. The device of any one of claims 22 to 26, wherein the actuator is an expandable member configured to apply the compressive force to the tumor or tissue when in an expanded state.

28. The device of any one of claims 19-27, wherein the device is formed from a flexible material configured to attach to an external skin surface at a proximate location from the tumor.

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