Cetuximab-Conjugated Per�uorohexane/Gold Nanoparticles for Low Intensity Focused Ultrasound Diagnosis Ablation of Thyroid Cancer Treatment

Chemotherapeutic e�cacy plays a signi�cant role in the development of nanotheranostic systems for drug delivery in tumor cells. In this study, we demonstrate the self-assembly of C225 conjugate, Per�uorohexane/Gold Nanoparticles (Au-PFH-NPs), which results in low-intensity focused ultrasound diagnosis ablation of thyroid cancer treatment. Cetuximab-Conjugated Per�uorohexane/Gold Nanoparticles (C-Au-PFH-NPs) showed excellent stability in water, PBS, and 20% rat serum. Transmission electron microscopy images revealed the effective construction of C-Au-PFH-NPs with commonly spherical assemblies. The incubation of C625 thyroid carcinoma with C-Au-PFH-NPs triggered apoptosis, which was con�rmed by �ow cytometry analysis. The C-Au-PFH-NPs showed remarkable antitumor e�cacy in human thyroid carcinoma xenografts. The histopathological results additionally con�rm the achieved outcomes. Furthermore, we successfully examined the e�ciency of C-Au-PFH-NPs when using the thyroid carcinoma low-intensity focused ultrasound (LIFUS) diagnostic imaging in vivo. These �ndings are clear for LIFUS agents with high performing images. It is also identi�ed that different therapeutic purposes will have extensive potential for future biomedical purposes.


Introduction
Anaplastic thyroid carcinoma (ATC) is one of the most malignantcarcinomas.It is comparatively rare, and is characterized by fastproliferation, neck invasion, and remote metastasis [1][2][3][4].ATC'ssevere prognosis is due to the rapid progression of tumors beforediagnosis.Current treatment is based on different combinations ofchemotherapy, and exterior ray radiation has been unsuccessful inenhancing survival, resulting in an average survival rate of 4 to 6months and less than 20% survival rate in 12 months [5][6][7][8].Therefore, there are convincing arguments for the development of anew theranostic approach for initial nding and e cient ATCtreatment [9][10][11].
Recently, triggerable drug-charged nanocarriers coupled withmultiple internal or external stimuli, such as pH, temperature,ultrasound, laser, and microwave radiation, have been extensivelyexplored for personalized treatment to enable controlled release.They have shown an excellent possibility to deliver enhancedanticancer treatment impact, with decreased systemic toxicity [12][13][14].Low-intensity concentrated ultrasound (LIFUS) has beenexhaustively researched for tumor treatment along with the use ofultrasound imaging analysis as a potential exterior activate, whichis noninvasive and displays signi cant tissue-penetratingcapacity.Particularly, it can signi cantly increase the e cacyof chemotherapy, avoiding harm to nearby cells, and reducingadversarial side effects [15].However, the discharge ofLIFUS-triggered drugs from nanocarriers and further tumor therapyis still unsatisfactory.This is largely attributable to thecomparatively lower accumulation e cacy of nanoparticles-chargednano transporters at the tumor sites.Accordingly, numerousnanotransporters have been extensively examined to enhance theaggregation of a large number of tumors without causing any sideeffects [15][16][17].
Several reports have shown that overexpression of the epidermalgrowth factor receptor (EGFR) is strongly associated with tumorprogression, migration, and invasion.EGFR is common in ATCpatients [18].
Antibodies or small molecules based on EGFRimmunotherapy can signi cantly increase the therapeutic effectagainst ATC.A human, murine chimeric EGFR-targeted monoclonalantibody called cetuximab has shown high empathy in the human EGFRextracellular domain.It inhibits the signals of the epidermalgrowth factor in cells by delaying usual receptor functions [19][20][21].The Food and Drug Administration (FDA) has approvedpreclinical treatments using cetuximab for the treatment of neckand head carcinoma and colorectal carcinoma with EGFR-expressingcancer tumors.This C225 might be a suitable objective for thestructure of nanocarriers to improve the outcome of ATC therapy.Remarkably, some researchers have revealed that for a wide spectrumof cancers, the blend of C225 with CPT-11 equivalents such asPer uorohexane/Gold Nanoparticles (Au-PFH-NPs) has signi cantsynergetic antitumor effects [22][23][24][25], thus, enhancing ATCdiagnostics.However, owing to the reduced vascular dispersal ofC225 and the hydrophobicity of the Au-PFH-NPs, the nanoparticles'(NPs) penetrability in the growth and their quantity in the tumorarea were inherently imperfect, which greatly debilitated theiranticancer e cacy.Fortunately, these problems can be diminishedby incorporating Au-PFH-NPs and C225 into one nanotransporter toattain a C225 and Au-PFH-NP combination chemotherapy whilesimultaneously enabling the targeted capability for nanocarriers [26][27][28][29].
Furthermore, monitoring medical imaging is essential for earlydiagnosis and tumor progression.
Numerous researchers have proposedthat LIFUS has the potential to achieve concurrent ultrasound (US)and medication transfer, meeting the present need of the initialtreatment and the ATC therapy [30][31][32].Due to variability and hugedimensions of microbubbles to realize the tumor, the theranosticstrategy of conservative US agents, such as microbubbles,demonstrate outstanding imaging capability but are not appropriatefor drug delivery.To avoid this problem, we intensively studiedphase-changing NPs that could be activated via LIFUS.Phase-changing NPs provide important bene ts in tumor theranosticsfor the supply of tumor US and US-triggered drugs [33][34][35].This newstrategy offers the possibility of developing a treatment formalignancy and addressing the present theranostic needs,signi cantly contradicting ATC.
The objective of this study was to modify the C225 nanocarrierto targetedly prevent ATC that might accrue in cancer cells, inaddition to the enhanced permeability and retention (EPR) effect,through the tumor homing belongings of the C225.The Au-PFH-NPspayload release and the LIFUS-triggered synergistic chemotherapywith C225 potentially make the best use of therapeutic e cacy,improving USI, and diminishing the side effects of chemotherapy, asshown in Figure 1.Due to its tremendousbiodegradability and biocompatibility, we used a per uorohexane(PHF core as the shell structure of the nanocarrier.We thensynthesized phase-changing NPs with PHF liquid (29°C boilingpoint).Meanwhile, the Au-PFH-NPs were burdened into the NPs whilethe C225 was conjugated on the surface of gold nanoparticles,affording (C-Au-PFH-NPs) C225-conjugated Au-PFH-NPs-charged phasetransformation.To our knowledge, this is the rst work of aLIFUS-mediated C225 modi ed nanosystem that assimilates tumorstargeting both US imagery and US activated drug conveyance totackle ATC.

Experimental Section
The detailed experimental procedures are given in the supportinginformation.

Cell culture and nude mice
The Cell Bank of the Chinese Academy of Sciences (Shanghai,China) acquired a human anaplastic thyroid carcinoma line (C643).The cells were grown in medium RPMI-1640 containing 10% FBS and 1%penicillin-streptomycin at 37°C in humidi ed air with 5%CO 2 .At the Laboratory Animal Center of Department ofUltrasound, Harbin Medical University Cancer Hospital (Harbin,China), BALB/C Female both mice and nude mice (balancing about 19g, 25 days) were bought then raised.All animals on our studieswere collected from the Harbin Medical University Cancer HospitalLaboratory Animal Center and retained in accordance with rulesauthorized by the Harbin Medical University's Animal EthicsCommittee (Harbin, China).Furthermore, all animal experimentalactivities were strictly in line with the policy of the HarbinMedical University's Institutional Animal Care and Use Committee(IACUC), and this study was endorsed by the IACUC.
In order to start an ATC model in nude mice, C643 cells werecollected, splashed thrice with the FBS free medium of RPMI-1640,and subcutaneously inoculated into each mouse's left ank (3× 10 7 C643 cells in 150 μL FBS freemedium of RPMI-1640 each mice).A Vernier caliper was used tomeasure the length and width of the tumour and the tumour quantitywas considered by the calculation: volume-(length aswidth× 2)/2.

Apoptosis examinations
The cells were seeded (4×10 6 C643 cells per well, 1.5mL) into a 6-well dish and grown at 37°C in a humidi ed incubatorwith 5% CO 2 for 24 hours.The IC 50 concentration used by Au-PFH-NPs and C-Au-PFH-NPs.The cellapoptosis assay grouping technique was in accordance with the cellviability assay group.After administering IC 50 concentration of the formulations of Au-PFH-NPs and C-Au-PFH-NPswas implemented 2 hours later [39][40][41].

Cell cycle arrestexaminations
The cells were seeded (4×10 6 C643 cells per well, 1.5mL) into a 6-well dish and grown at 37°C in a humidi ed incubatorwith 5% CO 2 for 24 hours.The IC 50 concentration used by Au-PFH-NPs and C-Au-PFH-NPs.The cells weregathered and analyzed in the PI-stained cells after 24 hours ofculture, and the percentages of the cells in the G0/G1, S phase,and G2/M phases were evaluated [42][43][44].

In vitro uorescence imaging inxenografts tumour
A continuous dosage of DiR labeled Au-PFH-NPs and C-Au-PFH-NPs(2 mg/mL, 200μL) was given to C643 tumour-bearing mice.With 1%pentobarbital, all mice were totally narcotized and uorescencepictures were acquired before injection and 3h, 6h and 24 hpost-injection.A vivid uorescence imaging for tiny animalsevaluated the uorescence intensity changes in the tumour areasin vivo.For ex vivo uorescence imaging, thesigni cant organs and tumour of one mouse were gathered.Inaddition, Dil-labeled Au-PFH-NPs and C-Au-PFH-NPs (2.5 mg per mL,150 μL) were injected through the intravenous of C643tumourbearing mice were injected six hours after injection.At thepredetermined post-injection moment, tumour matters and signi canttissues were gathered, segmented, and ice-covered.DAPI dyeing wasconducted in the dark for 5 min after fastening with 4%paraformaldehyde.The biodistribution of Dil-labeled Au-PFH-NPs andC-Au-PFH-NPs was monitored by CLSM [45][46][47].

Therapeutic e cacy of invivo
When the subcutaneous tumour reaches 100 mm 3 involume, an antitumour assay was conducted on xenografts of micecarrying anaplastic thyroid cancer.The tumour-bearing mice werearbitrarily split into 3 communities (n-5 per unit): control group(Saline) and free Au-PFH-NPs and C-Au-PFH-NPs were administered bythe organizations .Two hundred microliters of the blend wasinjected with the same dose of Au-PFH-NPs and C-Au-PFH-NPs (1mg/kg) through the tail vein in a 1% saline solution weredetermined six hours after injection with the US agent lling theinvestigation with the tumour super cial.Afterward theinoculations of C643 cells, 5 consecutive treatments were performedeach 72 hours starting on day 20 and ending on day 37.Each mouse'stumour dimensions and weight was recovered every three days, andchanges in tumour volume were examined from the relative tumourdimensions V/V 0 (V 0 : initial volume prior totreatment), and tumour growth curves were drawn at the same time.On day 37 days, all mice were euthanized and dissected and weighedthe tumour masses.In addition, studies in histology andimmunohistochemistry were conducted.Sections of the tissue werestained with histophalogy [48][49][50].

Characterization of C-Au-PFH-NPs
Having these compounds at hand, we examined the transmissionelectron microscopy (TEM) analysis of Au-PFH-NPs (1 and 1a zoomview) and C-Au-PFH-NPs (Figures 1 and2).Their ability to recapitulatethe self-assembly behavior in aqueous solutions was tested.Forthis purpose, we dissolved the C-Au-PFH-NPs prodrugs in dimethylsulfoxide (DMSO 10 mg/mL) and then rapidly injected them intodeionized (DI) water under ultrasonication.This procedure allowedus to validate that the solution was transparent and slightlybluish.Electron microscopy revealed that the drug moleculesself-assembled to form a spherical nanoparticle structure.Thedynamic light scattering (DLS) showed a single peak distribution ofthe nanoparticles.The average hydrodynamic diameter (intensity) ofthe compound 1 was ~107.1 nm, and compound 2 was ~108.0 nm(Figure 2B).There is, however, a certain adhesionbetween nanoparticles formed by the self-assembly of simplesmall-molecule drugs [51][52][53].Therefore, we have a miscible liquidwith many hydrophobic drugs by combining the prodrug with theappropriate amount of C225 molecules.These nano-assemblies wereformed and widely used for in vivo drug delivery, to solve theproblem of adhesion and to optimize cancer-speci c drug delivery.Then, we measured the stability of C-Au-PFH-NPs with variousparameters such as water, PBS, and 20% rat serum, which showed asigni cantly stable size in various parameters (Figure2C).Although C-Au-PFH-NPs can self-assemble to formnanoparticles, taken together they may not be su ciently stable.Therefore, C225 nanoparticles loaded with Au-PFH were investigatedfurther to evaluate their anticancer e cacy in vitro.

In vitro intracellularuptake
As illustrated in Figure 3, the much tougherred uorescence derived from Dil-labeled C-Au-PFH-NPs was moreconcentrated in the C-Au-PFH-NPs group around the cytomembrane ofC643 cells compared to the non-target and antagonistic groups [54].Furthermore, larger quantities of red uorescence were noted afterexposure to the C-Au-PFH-NPs group.These ndings indicated thatdue to the elevated tumor-homing characteristics of C225, theC-Au-PFH-NPs could adhere to C643 cells, and considerablyencouraged intracellular uptake of the C643 cells.In theresentment group, the C-Au-PFH-NPs lost the capacity to target theC643 cells because of the congestion of surplus free C225, leadingto low levels of C-Au-PFH-NPs around the cells.This demonstratedthat the C-Au-PFH-NPs' desired targeting effectiveness was theoutcome of the EGFR-mediated directing capacity.

In vitro cytotoxicity assay
The cell counting kit -8 assay assessed the cell viability ofdifferent NP formulations at distinct levels, through adose-dependent model.As illustrated in Figure 4A, the cellviability of nanoparticles in the analyzed dose range was greaterthan 80% at 10 mg/mL.The comparatively small and insigni cantviability proposed that the elevated biocompatibility ofphase-changing NPs was appropriate for in vivo application.Reasonably, the cell viabilities of Au-PFH-NPs and C-Au-PFH-NPsdecreased considerably as levels of C-Au-PFH-NPs increased.Particularly, the cell viability of the cells treated withC-Au-PFH-NPs was low at the same concentration, implying that themixture of C-Au-PFH-NPs could boost cytotoxicity synergistically.Cell viability of C-Au-PFH-NPs.The remarkably improvedcytotoxicity of C-Au-PFH-NPs may be due to the increased cellmembrane permeability caused by the cavitation effect and theimproved cell viability of C-Au-PFH-NPs at the objective location,signi cantly increasing the inhibitory impression of C-Au-PFH-NPson cell development.

Cell apoptosis and cell cycle assays
Next, we evaluated cellular apoptosis.In several groups, totalapoptosis (TA) improved as follows: control and Au-PFH-NPs andC-Au-PFH-NPs, respectively (Figure 4B).It should be noted that theapoptosis proportion of Au-PFH-NPs was smaller than that ofC-Au-PFH-NPs, whereas it was signi cantly greater than that ofC-Au-PFH-NPs.Cell cycle assays were also conducted to evaluatewhether the cell cycle was in uenced by the anti-proliferationused by Au-PFH-NPs and C-Au-PFH-NPs.Compared to the control group,a higher percentage of the G2/M phase was perceived in allpreserved sets (Figure 4C).The arrest cycle in the C-Au-PFH-NPsG2/M phase was higher than that in the control group but higherthan that in the C-Au-PFH-NPs, which is consistent with the resultsof cytotoxicity and the apoptosis assays mentioned above.Anenhanced proportion of the cell cycle in the G2/M phase wasdetected, and the distinct declaration stages were ascribed to thedistinct effects among distinct tumor cells.Hence, theC-Au-PFH-NPs showed an important cell cycle arrest in C643 cellsduring the G2/M phase impact.
Complete deliberation of the outcomes of the in vitro experimentindicated that nanoparticles could aid as exceptional vehicles forAu-PFH-NPs and C-Au-PFH-NPs.Their combination with C225 enabledthe targeted enhancement of cell recognition and endocytosis andenhanced the therapeutic effect of C-Au-PFH-NPs.Furthermore, wemaximized the cell proliferation inhibitory effects which may bedue to the increased cell membrane permeability caused bycavitation and ultrasound-targeted microbubble destruction (UTMD)effects improved the release of C-Au-PFH-NPs at the objective site,signi cantly increasing the inhibitory effect on proliferation ofthe tumor cells.
There wasvirtually no distinction between the targeted and non-targetedgroups in the intensi ed uorescence in the corresponding bodies.Meanwhile, considerably greater red uorescence signals were notedin the targeted group's tumor cryosections at 6 h under CLSMfollowing the tumor tissue's ultrathin segment, relative to thefewer red signals in the non-target group.It should be noted thatthe red uorescence signals in the tumor cryosections of theAu-PFH-NPs group were signi cantly better compared to those of theC-Au-PFH-NPs group after irradiation.The distribution of the uorescence signal in the main organs showed no signi cantalteration in either group, except in the liver and spleen (Figure5B and C).The non-targeted group's low uorescence signals at thetumor site may have resulted from the EPR effect, which facilitatedinert combination in tumor tissues.Contrastingly, the improved uorescence signal of the target group was primarily due to theC225-mediated endocytosis mechanism.Additionally, the C-Au-PFH-NPscould overcome the biological barriers of the tumor.
Theaccumulation of C-Au-PFH-NPs at the objective places was endorsedafter microbubble oscillation, cavitation, and destruction.Duringthe process of the oscillation and crash of the acousticmicrobubble by the US-targeted microbubble removal impact, the cellmembrane could be interrupted, and it's permeability was enhanced,allowing the greater accumulation of C-Au-PFH-NPs at the objectivelocations.These conclusions further con rm that C225 has thepotential to precisely carry nanocarriers to tumor cells,preventing them from rapidly reentering into the systemiccirculation, allowing extravascular diagnosis and e cientantitumor therapy with an agent.

In vitro ultrasound imaging
Based on the targeted accumulation capacity of Au-PFH-NPs andC-Au-PFH-NPs in tumor cells, we explored the potential ofphase-changing nanoparticles to aid as US contrast to improve USIand treatment scratches [55][56][57].Following the administration ofvarious medicines before LIFUS irradiation, even lower or anechoiccontrast improved US signals were observed in each group(Figure 6A).LIFUS was performed in all groups 6 hafter the administration of various treatments, at the same timeperiods with in vivo ultrasound imaging.Expressively sturdierspot-like echo signs slowly accrued in both modes at the tumorplaces in the treated group, while no evident deviations weredetected in the saline group, and only negligible signs appeared inthe non-target group.This suggested that C225 eased the directionof the tumor tissue accretion.Large quantities of microbubbleswere produced when phase-changing NPs were subjected to ADV at theLIFUS-triggered tumor site, resulting in improved US imaging.However, owing to the absence of C225-mediated targeting capacity,the inadequate ADV of the Au-PFH-NPs and C-Au-PFH-NPs could noteffectively improve ultrasound imaging.Furthermore, no obviousenrichment was observed without the LIFUS irradiation in theAu-PFH-NPs, and the C-Au-PFH-NPs alone could not improve theultrasound imaging in vitro, shown in Figure 6B-D.These ndings highlighted that C-Au-PFH-NPs were appropriate asultrasound imaging agents and e cient as in vivonanocarriers because of their relative stability.This is in linewith the outcomes of the ultrasonic imaging, additionally verifyingthe effectiveness of the beleaguered ultrasonic of C-Au-PFH-NPslower than that of the potential of LIFUS irradiation and localLIFUS radioactivity to boost the precision of phase-changingC-Au-PFH-NPs.

Therapeutic e cacy invivo
The antitumor e cacy in vivo was explored in thesubcutaneous C643 models, to assess the therapeutic e cacy of themixture of Au-PFH-NPs and C-Au-PFH-NPs in vitro.Numerouspictures of separate groups of mice were drawn to demonstrate theimpact of the therapy.(Figure 7A-C).Thetherapeutic effectiveness was evaluated by tracking changes in eachgroup's tumor volume.It was observed that the tumor in the salinegroups was debauched, and there was no signi cant decrease in thetumor dimensions in the C-Au-PFH-NPs group, indicating that itsdose was dependable in vivo, and that thewell-known epidermal growth factor was a target for tumor cellidenti cation and treatment.However, the C-Au-PFH-NP accumulationat the tumor site depended solely on the existence of vesselfenestration and vascular leakage, and the inadequate drug releaseat the tumor site restricted the therapeutic effect.These ndingsshowed that the C-Au-PFH-NPs in nude mice could enhance thetherapeutic effect of anaplastic subcutaneous thyroid cancer.Additionally, compared with the control (saline) groups, H&E,procaspase 9 (brown), and cleavedcaspase 3 (brown) expressionlevels were enhanced.The Ki67 staining and TUNEL assay were usedto measure the apoptosis of the tumor in vivo (Figure 7D).Furthermore, while Au-PFH-NPs signi cantly reduced body weightduring the course of the therapy, the use of C-Au-PFH-NPs showed nostatistically signi cant impact on the body weight among all micegroups.
The above ndings clearly highlight that the combination ofC-Au-PFH-NPs attained a notable excellent therapeutic effect tocounter ATC in nude mice, highlighting the importance of thesecurity of beleaguered tumor treatment.This diagnostic approachis preferred for ATC, signi cantly improving the healing capacity,without noticeable side effects.

Conclusion
The data presented in this study highlight a strategic rationalefor the effectiveness and safety of Au-PFA-NPs.As the syntheticAu-PFA-NPs and C-Au-PFA-NPs are fully biocompatible composites withminimal modi cations, the safety risks can be minimized byconsidering their clinical translation.Furthermore, consideringthe ability of Au-PFA-NPs to overcome the cetuximab (C225)-conjugated C-Au-PFA-NPs, it was expected that this approach couldbe an optional therapeutic platform to treat patients withdrug-resistant cancer.Lastly, we envision that in addition totaxane agents, this C-Au-PFA-NPs-based approach could be a simpleyet broadly applicable strategy to improve tolerance and present abetter organized cytotoxic nanotherapeutic approach compared toother antitumor agents.

Figure 2 Analysis
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Figure 5 In
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