Extravascular gelation shrinkage-derived internal stress enables tumor starvation therapy with suppressed metastasis and recurrence

Despite the efficacy of current starvation therapies, they are often associated with some intrinsic drawbacks such as poor persistence, facile tumor metastasis and recurrence. Herein, we establish an extravascular gelation shrinkage-derived internal stress strategy for squeezing and narrowing blood vessels, occluding blood & nutrition supply, reducing vascular density, inducing hypoxia and apoptosis and eventually realizing starvation therapy of malignancies. To this end, a biocompatible composite hydrogel consisting of gold nanorods (GNRs) and thermal-sensitive hydrogel mixture was engineered, wherein GRNs can strengthen the structural property of hydrogel mixture and enable robust gelation shrinkage-induced internal stresses. Systematic experiments demonstrate that this starvation therapy can suppress the growths of PANC-1 pancreatic cancer and 4T1 breast cancer. More significantly, this starvation strategy can suppress tumor metastasis and tumor recurrence via reducing vascular density and blood supply and occluding tumor migration passages, which thus provides a promising avenue to comprehensive cancer therapy.

growth method. In detail, 10 mmol CTAB was dissolved in 10 mL of HAuCl 4 solution (5 mM), and subsequently the solution was diluted into 100 mL with DI water and vigorously stirred. Afterwards, 120 μL of AgNO 3 solution (0.1 M) was dropwise added and the reaction proceeded for 5 min.
Immediately afterwards, 600 μL of ascorbic acid (AA) solution (0.1 M) was added above solution to reduce Au (III) to Au (I), followed by injecting 40 μL of NaBH 4 solution (0.1 M) to promote nucleation and growth, and the growth process lasted for another 4 h. The CTAB-capped GNRs were collected via high-speed centrifugation at 12000 rpm for 15 min. Furthermore, the obtained GNRs were repeatedly washed via dispersion in DI water under ultrasonication and centrifugation at 12000 rpm for 15 min.

Synthesis of PEG-coated GNRs (GNR-PEG-SH)
The surfaces of the as-prepared CTAB-capped GNRs were modified with thiol-modified PEG (HS-PEG-SH) via a classic ligand-substitution method. Typically, 2 mM K 2 CO 3 and 1 mM HS-PEG-SH aqueous solutions were added to the aforementioned CTAB-capped GNRs solution at a volume ratio of 1:0.2:10, respectively. After 24 h incubation at room temperature, GNR-PEG-SH was harvested and repeatedly washed via dispersion in DI water and high-speed centrifugation under at 12,000 rpm for 15 min. Ultimately, the collected GNR-PEG-SH was dispersed into DI water and kept at 4 °C for use. The absorbance of the GNR solution was measured using a Shimadzu 3101PC UV/Vis spectrophotometer. The atomic concentration of GNRs was quantified by an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Agilent). FETEM (field emission transmission electron microscopy) analysis was conducted with a JEM 2100 F electron microscope operated at 200 kV to characterize the PEG-coated GNRs. Scanning electron microscopy (SEM) images/scanning transimission electron microscopy (STEM) images and corresponding element mapping were obtained on a field emission Maggellan 400 microscope (FEI Company).

Synthesis of linear pNIPAAm-co-AAc coploymers
Linear pNIPAAm-co-AAc copolymers were yielded via a well-established in-situ free radical copolymerization method using NIPAAm and AAc as the raw materials. Typically, in a three-necked, round-bottomed flask, 44.25 mmol of NIPAAm was dissolved in 90 mL of methanol along with a certain amount of AAc in sequence, followed by with ultrasonic agitation for 5 min. The polymerization that was accelerated by AIBN initiator was carried out under N 2 atmosphere at 60 °C and lasted for 24 h, during which continuous stirring was always accompanied. Afterwards, the resultant copolymers were harvested via the solvent extraction-mediated precipitation method after adding excessive diethyl ether, and subsequently they were further rinsed by dissolution in acetone and precipitation in excessive diethyl ether for three cycles. Ultimately, the obtained pNIPAAm-co-AAc copolymers were dried in vacuum at room temperature for 24 h prior to further use.

Synthesis of chitosan/mPEG-mal/pNIPAAm-co-AAc hydrogel and its derivative (hydrogel-GNR)
Briefly, chitosan/mPEG-Mal/pNIPAAm-co-AAc composite hydrogel (0.9 g) at the feed ratio of 20/10/70 was dissolved in 70 mL of acetic acid solution (2% v/v). The solution was shaken for 2 days and was then filtered using a glass funnel to remove undissolved materials, and the ultimate solution was stored at 4 °C for use. As for hydrogel-GNR, GNRs were added in above initial mixture and stirred for 8 h so as to guarantee the chelation of GNRs via the thiol-maleimide click reaction between GNR-EPH-SH and mPEG-Mal. Ultimately, the obtained composite solution was stored at 4 °C for use. The pores of as-prepared gel were characterized by scanning electron microscope (SEM) on a field-emission JEOL JSM-6700F microscope. The absorbance of the hydrogel-GNR sol was measured using a Shimadzu 3101PC UV/Vis spectrophotometer.

Synthesis of hollow porous Fe 3 O 4 nanoparticles
The magnetic porous hollow Fe 3 O 4 nanoparticles were obtained according to a simple solvothermal reaction. In brief, FeCl 3 ·6H 2 O (1.350 g) was completely dissolved in 70 mL of EG by magnetic stirring and ultrasonic irradiation, and then NH 4 Ac (3.854 g) was added into above mixture for further dissolution.
The above mixture was dissolved completely under vigorous magnetic stirring at room temperature for 30 min, and then was transferred into a teflon lining of hydrothermal synthesis reactor (100 mL capacity) and sealed by high pressure-tolerant outer lining made of stainless steel. Afterwards, the high pressure reactor was incubated in a preheated oven at 200 °C for 16.5 h. After cooling the reactor to room temperature, the black HIONs were collected and washed with deionized water, respectively. Size and structure were analyzed via Scanning electron microscopy (SEM) on a field emission Maggellan 400 microscope (FEI Company) and transmission electron microscopy (TEM) on a JEM-2100F electron microscope operated at 200 kV were N 2 desorption and adsorption isotherms, Zeta potential, XRD patterns.