Acta Pharmaceutica Sinica B 2023;13(4):1711e1725

Chinese Pharmaceutical Association

Institute of Materia Medica, Chinese Academy of Medical Sciences

Acta Pharmaceutica Sinica B

w w w. e l s ev i e r. c o m / l o c a t e / a p s b
w w w. s c i e n c e d i r e c t . c o m

ORIGINAL ARTICLE

Self-assembled multifunctional nanotheranostics

against circulating tumor clusters in metastatic
breast cancer
Ramya Dhandapani, Swaminathan Sethuraman,
Uma Maheswari Krishnan, Anuradha Subramanian*

Centre for Nanotechnology & Advanced Biomaterials, School of Chemical & Biotechnology, SASTRA Deemed
University, Thanjavur 613401, India

Received 12 August 2022; received in revised form 19 October 2022; accepted 25 November 2022

KEY WORDS Abstract Circulating tumor clusters (CTC) disseminating from the primary tumor are responsible for
secondary tumor formation where the conventional treatments such as chemotherapy and radiotherapy
Self-assembly;
does not prevent the metastasis at locally advanced stage of breast cancer. In this study, a smart nanother-
Hybrid nanotheranostic
system;
anostic system has been developed to track and eliminate the CTCs before it can colonize at a new site,
Circulating tumor clusters; which would reduce metastatic progression and increase the five-year survival rate of the breast cancer
Advanced breast cancer; patients. Targeted multiresponsive (magnetic hyperthermia and pH) nanomicelles incorporated with
Cancer stem cells; NIR fluorescent superparamagnetic iron oxide nanoparticles were developed based on self-assembly
Heterogenous clusters; for dual modal imaging and dual toxicity for spontaneous killing of CTCs in blood stream. A heteroge-
SPIONs; nous tumor clusters model was developed to mimic the CTCs isolated from breast cancer patients. The
NIR; nanotheranostic system was further evaluated for the targeting property, drug release kinetics, hyperther-
Metastasis; mia and cytotoxicity against developed CTC model in vitro. In vivo model in BALB/c mice equivalent to
CD44 stage III and IV human metastatic breast cancer was developed to evaluate the biodistribution and ther-
apeutic efficacy of micellar nanotheranostic system. Reduced CTCs in blood stream and low distant organ
metastasis after treatment with the nanotheranostic system demonstrates its potential to capture and kill
the CTCs that minimize the secondary tumor formation at distant sites.

ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical
Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

*Corresponding author.
E-mail address: anuradha@bioengg.sastra.edu (Anuradha Subramanian).
Peer review under responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

https://doi.org/10.1016/j.apsb.2022.12.003
2211-3835 ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting
by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1712 Ramya Dhandapani et al.

1. Introduction polymer with glucosamine repeating units and shows pH-responsive

characteristics on protonation of amine groups18. Low molecular
Chemotherapy and radiotherapy-induced dissemination of tumor weight chitosan grafted with oleic acid have induced fibrosis around
clusters in breast cancer promotes secondary metastases at distant primary breast tumor in mice, thereby inhibiting tumor progres-
organs, thereby worsening the quality of life and survival rate of sion19. Nanocarriers of medium molecular weight chitosan
patients at locally advanced stage1,2. Circulating tumor clusters (>150 kDa) have shown improved circulation duration and lesser
(CTCs) possess 50 times higher metastatic potential when compared solubility than low molecular weight chitosan in physiological
to single circulating tumor cells (sCTC). This results in enhanced media, which provides greater stability under systemic circulation20.
engraftment and colonization of clusters that would develop into Therefore, in this study, combinatorial treatment modalities
secondary tumors at distant organs3,4. Only 2.6% of overall sCTC have been integrated with specific targeting towards CD44-
population carry tumor initiating potentials expressing stem-cell expressing CTCs to track, trap and eliminate them from sys-
like markers such as CD44 and CD1335. Further, eliminating temic circulation. Subjecting CTCs to magnetic hyperthermia
CTCs from the blood before secondary colonization is a challenge would sensitize the tumor cells towards chemotherapeutic drugs
due to its shorter circulatory life span (up to a few h), lack of specific thereby increasing their cytotoxic potential. Nanocarriers were
targets with varied degrees of cellular heterogeneity, and tumor- designed to prevent non-specific internalization in normal cells by
forming ability6. Hence, advanced therapeutic and diagnostic ap- incorporating multi-stimuli responsive elements (pH and temper-
proaches with high specificity towards CTCs in addition to longer ature) and CD44-receptor targeted ligands. To achieve this, me-
circulation duration and spontaneous cellular internalization with dium molecular weight chitosan chains were grafted with lauric
stimuli-responsive drug release become indispensable to block cell acid to fabricate amphiphilic chitosan to self-assemble into
cycle process and induce apoptosis during systemic circulation. In nanomicelles. We hypothesize that conjugating with lauric acid
addition, nanocarriers with poor physical and chemical stability would enable thermoresponsive behavior to impart burst release in
under hemodynamic shear stress can lead to rupture of nanocarriers nanomicelles, which promotes spontaneous cell death of CTCs.
and non-specific release of drug in circulation7. Thus, developing However, specific trapping and killing of CTCs can be achieved
nanotheranostics would be challenging as it requires high degree of by conjugating CD44 antibodies on the micellar nanotheranostic
targeting specificity in circulation to facilitate both diagnostics and particles. Additionally, crosslinking chitosan with hexametaphos-
therapeutics for successful CTC targeted treatment strategy. The phate (HMP) or tripolyphosphate molecules would enhance the
major problem in CTC therapy is slow internalization into specif- structural stability of nanomicelles. Hence, lauric acid grafted
ically targeted CTCs under dynamic circulation, which can lead to chitosan-based micellar nanotheranostic systems are engineered to
poor cell killing ability of nanotheranostics. Clinical reports have encapsulate hydrophobic drug-loaded fluorescent iron oxide
shown higher recurrence rate and poor progression-free survival in nanoparticles (DFIO) with enhanced structural stability, stealth,
triple-negative breast cancer (TNBC) patients with higher number loading content, responsiveness, and targeting efficiency. In vitro
of CTCs8. Unfortunately, number of CTCs in blood and metastasis and in vivo efficacy was evaluated for the developed targeted
was amplified in TNBC patients undergoing radiotherapy and nanomicelles for identifying the adjuvant therapy and diagnostics
chemotherapy as their treatment regime tends to disintegrate the potential against CTCs.
tumor clusters rather killing completely. Hence, rapid internaliza-
tion and combinatorial treatment strategies would promote com-
2. Materials and methods
plete apoptosis in clusters of tumor cells and prevent recurrence of
tumors9. This would potentially inhibit occurrence of drug resis-
tance and prevent successful colonization of incompletely killed 2.1. Materials
tumor cell clusters10,11.
Matrix-based drug delivery systems cannot entrap other inor- Medium molecular weight chitosan (SigmaeAldrich, India), lauric
ganic entities due to minimum storage space and also lack constant acid (Himedia, India), EDC, bifunctional PEG (Jenkem Technology,
drug release over time based on the diffusion of a matrix12,13. Ve- USA), anti-CD44 mAb antibody (Novus Biologicals, USA), 4T1
sicular nanocarriers such as exosomes, polymersomes, micelles, cells, tumor-derived mammary endothelial cells (Cell Biologics,
dendrimersomes, and liposomes are advantageous over nano- and India), acridine orange, propidium iodide, Sytox blue nucleic acid
micro-particles, nanobullets because of their self-assembling nature stain, crystal violet and tubulin tracker (Molecular Probes, India)
and high encapsulation efficiency of hydrophobic cytotoxic moi- were used in the study. Traut’s reagent, 20 ,70 -dichlorofluorescin
eties13. Liposomes and micelles are clinically approved nano- diacetate (DCFDA), Ellmann’s reagent [5,5-dithiobis (2-
vesicular systems that enhance solubility and circulation duration of nitrobenzoic acid), DTNB], were purchased from SigmaeAldrich,
poorly soluble drugs in biological fluids leading to efficient phar- India.
macological and pharmacokinetics14. Micellar nanostructures have
shown various advantages including improved stability in biological 2.2. Synthesis of amphiphilic chitosan-g-lauric acid
fluids, entrapment of hydrophobic moieties, ease of surface func-
tionalization, and integrating active targeting moieties15. Ultra-low Chitosan (0.1% w/v) in 1% acetic acid solution was stirred overnight
critical micelle concentration (CMC) micelles have shown greater followed by the addition of EDC and lauric acid in the ratio of 16:1 to
stability in serum even at 10
2 mmol/L concentrations when conjugate lauric acid to the free amine groups of chitosan. After 24 h,
compared to existing polymeric micelles16. However, the stability of chitosan was precipitated using isopropyl alcohol and ammonia
micelles can be improved by introducing zwitterionic complexes to solution and washed with isopropyl alcohol (IPA) to remove
inhibit the dissolution of micellar structures during systemic circu- unreacted lauric acid. The chitosan solution was dialyzed against
lation. Stimuli-responsive polymers grafted with fatty acids like water to remove unreacted EDC and vacuum dried to obtain
stearic acid and palmitic acid have shown lower CMC values and amphiphilic chitosan-g-lauric acid (CS-g-LA). Critical micelle
higher loading efficiencies17. Chitosan is a widely used natural concentration of micelles was estimated by using pyrene as a
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1713

fluorescence probe after equilibration with polymer solution for confocal microscopy (FV1000, Olympus, USA). In the subsequent
24 h. CMC value was extrapolated using the intensities recorded at experiments, clusters were transferred to fresh plates and dosed
339 and 374 nm emission wavelengths for different concentrations with nanotheranostic particle suspension (Dox equivalent in
of amphiphilic polymer. 25 mg/mL) for 24 h before analysis. For hyperthermia treated
groups, clusters were transferred to eppendorfs and subjected to
2.3. Synthesis of magnetic chitosan-g-lauric acid micelles hyperthermia at 480 kHz for 5 min and stained with specific dyes
and antibodies for assays.
Drug conjugated superparamagnetic iron oxide nanoparticles
(DFIO) were prepared as mentioned elsewhere21. Magnetic chi- 2.5.3. Cytotoxicity assay
tosan-g-lauric acid (CS-g-LA) micelles were prepared by the Heterogenous tumor cell clusters (n Z 30) were treated with
dropwise addition of drug-conjugated superparamagnetic iron different treatment groups and incubated for 24 h, and Alamar
oxide nanoparticles (DFIO) solution into the amphiphilic chito- blue assay was performed to quantify the cytotoxic effect of the
san-g-lauric acid (CS-g-LA) solution under ultrasonication for developed nanomaterials against the cell clusters. Clusters were
10 min with 30% amplitude and 30s/30s pulse. Further, 0.5% stained with acridine orange and propidium iodide to visualize the
HMP was added to stabilize the micelles followed by the centri- apoptotic and dead cells in the clusters, respectively, using
fugation to obtain magnetic nanomicelles. confocal microscopy.

2.4. PEGylation and antibody tagging of magnetic nanomicelles 2.5.4. Internalization studies
Uniform-sized clusters were blocked with anti-CD44 mAb anti-
Stealth characteristic was introduced by conjugating heterofunc- body (Novus Biologicals, USA) in serum-free medium for 2 h
tional maleimide-PEG-NHS [NHS-PEG(2000)-Mal] over posi- prior to treatment with free Dox and Dox-loaded nanomicelles for
tively charged amine groups of chitosan on micelles. The degree 4 h. Clusters treated with nanomicelles without blocking CD44
of substitution for PEGylated nanomicelles was estimated using served as control. Samples were characterized using a laser
TNBS assay. avb3 Integrin and CD44 antibodies were thiolated scanning confocal microscope, and intensity quantification was
using Traut’s reagent for direct conjugation with free maleimide done using Image J software. Prussian blue staining was carried to
groups of PEG chains on magnetic nanomicelles. Thiolation stain iron deposition in the clusters using the protocol mentioned
percentage and binding efficiency of antibodies were estimated elsewhere22.
using Ellmann’s assay using standard plot of concentration vs.
absorbance for L-cysteine. 2.5.5. Mitochondrial membrane potential
Heterogenous tumor cell clusters were treated with free Dox and
2.5. In vitro evaluation of CD44-nano against heterogenous Dox-loaded nanomicelles for 24 h and mitochondrial depolarization
CTC model was evaluated in the presence and absence of magnetic hyperthermia
using carbocyanine dye JC-1. The treated cells were incubated with
2.5.1. Cell culture 10 mg/mL of JC-1 dye as per the manufacturer’s protocol for 30 min
4T1 cells were cultured with RPMI medium supplemented with and analyzed using Confocal Laser Scanning Microscopy (CLSM)
10% FBS and 1% PS and trypsinized at 80% confluency using with separate excitation sources for J monomer and J aggregate at 488
0.05% trypsin. Tumor-derived endothelial cells (mammary fat and 535 nm, respectively. The intensity was quantified using Image J
pad) were cultured with specific endothelial growth medium (Cell software and intensity ratio of red/green fluorescence was calculated
Biologics, USA) and trypsinized using 0.25% trypsin at 70%e and represented in bar graph.
80% confluency. Mammary fibroblasts were isolated from mam-
mary fat pad using enzymatic digestion method as mentioned 2.5.6. Anti-colonization assay
elsewhere and cultured in DMEM medium supplemented with Heterogenous clusters were transferred from non-adhesive surface
10% FBS and 1% PS. Cells were trypsinized using 0.05% trypsin to tissue culture polystyrene (TCPS)-treated confocal dishes and
at 80% confluency. All the cells were maintained in humidified were treated with free Dox and Dox-loaded nanomicelles. After
chamber with 5% CO2 at 37  C. 4T1 cells, endothelial cells and 24 h, the clusters were stained with Sytox blue to stain nuclei of
primary mammary fibroblasts of passage 12e20, 4e10 and 1e5 dead cells and images were captured in confocal microscopy. The
were used for experiments, respectively. total area of the extension was calculated by subtracting total area
covered by cells after 24 h from area of the initial cluster using
2.5.2. Developing heterogenous circulating tumor clusters Image J software images. Also, the diameter of CTC was
in vitro model measured using an inverted microscope.
4T1 cells (murine metastatic breast cancer cell line), tumor-
derived endothelial cells and mammary fibroblasts in the ratio of 2.6. In vivo biodistribution and therapeutic study in CTC model
1:3:2, respectively, were co-cultured in ultra-low attachment U
bottom 96-well plates up to 48 h to develop heterogenous CTCs. Female BALB/c mice (6e8 weeks) were procured from M/s Vivo
The clusters were collected using 100 mL of micropipette for Biotech Pvt Ltd., Hyderabad with Institutional Animal Ethics
further assays. Presence of CTC specific markers such as E-cad- Committee (IAEC) approval (567/SASTRA/IAEC/RPP). Six an-
herin, CD44 and CK19 were confirmed in the heterogenous CTCs imals were housed in a cage and fed ad libitum, followed by daily
through immunostaining technique. Rat anti-CD44 mAb and observation in a pathogen-free environment.
rabbit anti E-cadherin (Novus Biologicals, USA) antibody tagged
with Alexafluor-647 conjugated anti-rat IgG (Cell Signaling 2.6.1. Biodistribution study
Technology, USA) and Alexafluor-488 anti-rabbit IgG antibody Distribution of targeted and untargeted (5 mg/kg of ICG equiva-
(Molecular Probes, India). Images were taken using laser scanning lent) nanoparticles was administered through intravenous injection
1714 Ramya Dhandapani et al.

in BALB/c mice (6e8 weeks) was studied. Animals (n Z 3) were 2.7. Statistical analysis
anesthetized using ketamine/xylazine and placed inside the im-
aging chamber with warm air delivery to maintain the animal Statistical analysis was performed using SPSS 15.0 for Windows
temperature in In vivo Xtreme (Bruker, Belgium). The animals (SPSS v.15, USA). Values were represented as mean  standard
were simultaneously excited at 790 nm and subjected to X-ray error. Student’s t-test and one-way ANOVA was performed for
irradiation at various time points without disturbing the animal analyzing significant differences in value of means between
until the completion of experiment. Free Dox (5 mg/kg) distri- groups. The values were considered to be significant at P < 0.05.
bution (Ex-490 nm, Em-530 nm) was compared with nano-
formulations in animals to understand the cardiotoxic behavior of
developed nanotheranostics.
3. Results and discussion
2.6.2. Development of in vivo CTC model in immunocompetent
mice Conditions for engineering amphiphilic polymers for formation of
In vivo model was developed by intravenously injecting in vitro nanomicellar structures are influenced by various factors such as
heterogenous CTCs in two different densities (low
30; high molecular weight of polymer, hydrophilic to hydrophobic ratio,

90) through tail vein in BALB/c mice. Behavioral changes in the and critical micelle concentration. Polymers with low CMC
animals were monitored and body weight was recorded every (<0.1 mg/mL) possess extended circulation duration with greater
alternate day. After 16 days, the blood was collected and animals stability in blood and other biological fluids. Hydrophobic content
were sacrificed. Lungs were stained with Bouin’s stain to visualize in the amphiphilic polymer influences the loading content and
tumor nodules. Blood was treated with 6-thioguanine and further encapsulation efficiency of the hydrophobic drug within nano-
immunostained with CD44 antibodies to track circulating tumor micelles. Leakage of the drug can be minimized by increasing the
clusters in circulation. hydrophobic moiety in the amphiphilic polymer and prevent
dissociation of nanomicelles during circulation. Higher substitu-
2.6.3. Therapeutic study tion of hydrophobic groups results in slower drug release from
Preclinical testing of the developed magnetic nanotheranostics nanomicelles. Therefore, grafting of hydrophobic groups to a
was carried out by intravenous administration of the low-density hydrophilic polymer is challenging in terms of developing nano-
CTCs followed by nanotheranostics with equal concentrations of micellar structure with high drug-loading efficiency, long circu-
Dox in different groups. Animals (n Z 6) were administered with lation time, high stability in fluids, leak-proof and controlled drug
dosage once a week and subjected to hyperthermia weekly twice release, and improved tissue penetration. In this work, medium
at 480 kHz for 20 min. Body weights were recorded every alter- molecular weight chitosan was modified with lauric acid to induce
nate day and any behavioral changes were observed. After 18 amphiphilicity in chitosan chains (Fig. 1). Amphiphilic chitosan
days, animals were sacrificed and blood was collected for colony- can self-assemble into nanomicellar structures encapsulating
forming assay. Lungs were stained with Bouin’s stain to visualize DFIO in the hydrophobic core to target and kill circulating tumor
secondary tumor nodes, and other organs were fixed in formalin cells. Nanomicelles were engineered to possess stimuli-responsive
for histopathological evaluation. spontaneous drug release in the target cancer cell clusters in blood
circulation and eliminate before colonization at secondary tumor
2.6.4. Blood clonogenic assay
Blood collected from animals was cultured in vitro under sterile
conditions to estimate CTCs in systemic circulation that could
potentially induce secondary tumor sites at distant organs. Blood
was centrifuged at 8000 rpm to separate plasma, and the pellet was
washed several times in PBS after RBC lysis. Pellets were sus-
pended in RPMI medium with 60 mmol/L 6-thioguanine and 10%
FBS and transferred to 100-mm culture plates. The plates were
incubated at 37  C in an incubator with 5% CO2 for 5 days
without any media change. The unattached cells were removed by
subsequent washing with PBS. The colonies were fixed for 30 min
in methanol and stained with 0.5% crystal violet for 30 min.
Further, the stained colonies were viewed under a microscope and
the number of colonies were counted for each group.

2.6.5. Histopathological evaluation

The organs fixed in 10% buffered formalin solution were pro-
cessed using an automatic tissue processor (Leica TP1020) fol-
lowed by subjecting to paraffin embedding using a tissue
embedding station (Leica EG1150C). Tissue sections of 3-mm
thickness were cut from the paraffin blocks using a microtome
(Leica RM2125RTS) and affixed to super frost microscopy slides
to stain cells and connective tissue with hematoxylin and eosin
staining. For Prussian blue staining, the Prussian stain (1:1 po-
tassium ferricyanide: HCl) was added to tissue sections and Figure 1 Scheme for synthesis of amphiphilic chitosan-g-lauric
incubated for 20 min to visualize the iron deposition in the tissues. acid.
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1715

sites. Stimuli-responsive drug release facilitates enhanced intra- 20.15 mV recorded for the nanomicelles in an aqueous medium
cellular Dox delivery and cytotoxicity. can be contributed to high colloidal stability.

3.3. Synthesis of DFIO-loaded chitosan-g-lauric acid

3.1. Modification of chitosan-g-lauric acid (CS-g-LA) nanomicelles
Lauric acid grafting on chitosan chains (CS-g-LA) was mediated
Nanomicelle formation induced by self-assembly of amphiphilic
by EDC crosslinking for amide bond linkage between the amino lauric acid grafted chitosan using ultrasonication forms the nano-
group of chitosan and carboxyl group of lauric acid and was carrier for developed nanotheranostic agent. Hydrophobic DFIO
confirmed using FTIR and 13C-NMR spectra (Fig. 2a and (doxorubicin complex FIO) component during self-assembly gets
Supporting Information Fig. S1). Different EDC:LA ratios showed encapsulated in the core of the micelles to form core-shell nano-
variation in the integration of lauric acid with chitosan as observed structure. Iron content estimation using Prussian blue assay showed
in -CH stretch from 2865 to 3050 cm
1 (Fig. S1a), however at higher iron concentration with increasing concentration of DFIO
1:16 ratio a sharp alkyl eCH stretch at 2935 cm
1 in CS-g-LA (Table 1). However, nanomicelles showed distorted structures above
confirms the alkyl chains of lauric acid integrated into chitosan. 0.05:1 ratio (data not shown). Magnetic nanomicelles (MN) of
Also, the degree of substitution for lauric acid to chitosan was average 145.7  8.9 nm was obtained at the ratio 0.05:1 of iron to
estimated to be 17.05  0.9% by measuring the free amine groups polymer ratio (w/w), and a surface charge of 15.4 mV was recorded.
using 2,4,6-trinitrobenzenesulfonic acid as mentioned else- The size of nanomicelles increased after encapsulation of DFIO,
where23. Further, solid state 13C-NMR spectra for CS-g-LA where TEM and SAED data revealed aggregated nanostructure with
showed additional C-C peaks between 0 and 40 ppm similar to
dense crystalline DFIO entangled within polymeric chains similar to
lauric acid spectra and splitting of peak at 172 ppm could be bare iron oxide nanoparticles21 (Fig. 2c). Iron content was estimated
correlated to the amide bond formation between chitosan and to be 2.73  0.07 mmol/L/mg of MN. Inorganic content was esti-
lauric acid on grafting (Figs. 2a, S1b and S1c). mated to be around 44.3% in the DFIO-loaded nanomicelles (MN)
using thermogravimetric analysis in agreement with Prussian blue-
3.2. Preparation of chitosan-g-lauric acid micelles based iron quantification (Supporting Information Fig. S3d).

Critical micelle concentration using pyrene as a fluorescent probe 3.4. PEGylation and antibody conjugation
for CS-g-LA was estimated to be 0.625 mg/mL (Fig. 2b). Micellar
self-assembly was induced fabricated by ultrasonication of CS-g- Stealth characteristics was introduced in the nanomicelles by
LA at various concentrations, however at 1% (w/v), spherical conjugating heterofunctional maleimide-PEG-NHS over positively
polymeric nanomicelles were observed using FE-SEM charged amine groups of chitosan on MN which was confirmed
(Supporting Information Fig. S2beS2d). Further FE-TEM anal- using FTIR spectroscopy (Fig. S3a). FTIR spectrum of the PEGy-
ysis revealed hollow nanomicelles of average particle size of lated micelles shows the Fe-O stretching, C-N stretching of aliphatic
140  2 nm (Fig. S2e and S2f) and zeta potential value of amines, N-H bending and C]O stretch of chitosan at 522, 1079,

Figure 2 (a) NMR spectra of chitosan-g-lauric acid at EDC:LA ratio of 1:16, (b) I394/I363 vs. log of CS-g-LA concentration; (c) Transmission
electron micrographs (scale bar 50 and 20 nm) and (c) SAED pattern of magnetic nanomicelles (MN).
1716 Ramya Dhandapani et al.

3.7. Dual diagnostic potential

Table 1 Iron content in varying iron to polymer ratio.
Iron to polymer Iron content in Developed targeted nanotheranostics showed transverse relax-
ratio (w/w) nanomicelles (%)
ivity of 33.08 Fe 1/(mmol/L) 1/s and concentration-dependent
0.01:1 25.6 negative contrast property of the SPIONs encapsulated in
0.03:1 15.2 nanomicellar systems (Fig. 3e and f). The NIR property of
0.05:1 48.8 nanotheranostics showed concentration-dependent increase in
0.07:1 54.2
intensity, which can track small cell clusters even under deeper
tissues. Integration of ICG on HIO can be evidenced by the
1532, and 1636 cm
1, respectively. The degree of substitution for increased fluorescence intensity with respect to increasing iron
PEGylated MN was estimated to be 79.2  2.3% using TNBS concentration. This proves the complex formation of ICG and
(trinitrobenzene sulfonate) assay. Integrin and CD44 antibodies HIO is stable to improve sensitivity in CTCs colonization in
were thiolated using Traut’s reagent for direct conjugation with free deeper tissues with dual-modal imaging. This dual-modal im-
maleimide groups of PEG chains on MN. Thiolation efficiency of aging property of nanotheranostics proves potential tracking of
CD44 and integrin antibodies were estimated to be 96.5  2.3% and CTCs even under circulation and visualization of secondary
98  4.8%, respectively, using Ellmanns’s assay. However, thiolated localization in distant organs.
antibodies against CD44 and integrin were conjugated on MN with
an efficiency of 74.93  2.69% and 75.44  12.01%, respectively. 3.8. Cytotoxic potentials of nanotheranostic evaluated using
Magnetic properties confirmed superparamagnetic behavior of the in vitro CTC model
nanomicelles, however, the saturation magnetization of CD44-MN
was reduced to 30.5 emu/g (Fig. 3a). However, this decline did not 3.8.1. Receptor-mediated CTC internalization
affect the magnetic hyperthermia properties where the Heterogenous CTCs treated with CD44-magnetic nanomicelles
concentration-dependent SAR value of MN was estimated to be (CD44-MN) showed preferentially higher localization of Dox in
97.7  1.4 W/g at 480 kHz (Fig. 3b). CTCs than with avb3-targeted nanotheranostics (Fig. 4a), which
can be correlated to the higher expression of CD44 in CTCs
facilitating receptor-mediated internalization. This confirms the
3.5. Multi-responsive nanomicellar systems
efficiency of CD44-targeted nanotheranostics over avb3-targeted
nanotheranostics in targeting the CTCs. However, to validate the
The multi-responsive drug release property of the targeted mag-
CD44 specific uptake of the developed targeted nano-
netic nanomicellar systems (CD44-MN) minimizes the side ef-
theranostics, CTCs were blocked with anti-CD44 antibodies to
fects and toxicity to normal cells caused by free doxorubicin. The
mask the CD44 receptors in CTCs. Further, blocked CTCs were
presence of protonated amine groups in chitosan induces swelling
treated with CD44-nanotheranostics and showed the negligible
of nanomicelles at lower pH, which was evaluated in MN. Drug
presence of Dox in the cells when compared to the unblocked
release profile showed burst release of w72% of doxorubicin
CTCs (Fig. 4b). Therefore, a significant reduction in the in-
within 3 h at pH 5.2. However, at pH 7.4 there was significantly
tensity of Dox in blocked CTCs confirms CD44-mediated
lowered drug release (w28%) as shown in Fig. 3c. Due to its
internalization of the nanotheranostic systems, which can be
lower melting point, CS-g-LA, showed thermoresponsive
used for targeting cancer stem cells-like cells. However, free
behavior, which was evaluated by subjecting the nanotheranostic
Dox diffusion was observed in CTCs even after blocking their
particles to alternating magnetic field where the encapsulated
CD44 receptors using specific antibody and confirmed its toxic
superparamagnetic iron oxide nanoparticles (SPIONs) induce heat
side effects due to its non-specific diffusion. Further, Prussian
due to Neels Losses and Brownian motion. A stepwise release
blue staining was carried out to observe the accumulation of iron
profile was observed on subjecting the nanotheranostic particles to
in CTCs, which will facilitate the use of hyperthermia as adju-
alternate ON-OFF cycles of hyperthermia (Fig. 3d). On subjecting
vant therapy in addition to the NIR fluorescence for tracking
to hyperthermia, w18% of Dox was released initially, but no
(Fig. 4c).
cumulative dox release was estimated during the OFF cycle.
Similar trend was observed during the subsequent ON and OFF
3.8.2. Cytotoxicity profile
cycles of magnetic hyperthermia. This proves the temperature-
CD44-MN has been designed to integrate NIR fluorescent iron
responsive release of Dox from nanocarrier. Interestingly, the
oxide nanoparticles and further conjugation of bifunctional-PEG
reduction in saturation magnetization did not affect the potential
to improve the stealth of the micellar structure for longer systemic
of nanoparticles for hyperthermia-mediated therapy.
circulation. Cytotoxicity of the developed nanostructures and the
effect of hyperthermia in cell killing was evaluated. The micellar
3.6. Hemocompatibility nanocarrier is responsive towards low pH and high temperature,
which can enhance the pH and temperature responsive drug
One of the most important properties of nanotheranostic targeted release kinetics leading to spontaneous cell death. The tempera-
against CTCs is to possess hemo-compatibility as it has direct ture responsiveness was confirmed by treating CTCs with drug-
contact with the blood and blood components. Nanotheranostics loaded fluorescent iron oxide nanoparticles IO (DFIO), DFIO-
showed minimal hemolysis percentage (<2%) under acceptable loaded micelles, PEG-coated DFIO-micelles (PEG-MN), CD44-
threshold even at higher concentrations (1 mg/mL). Also, the MN in the presence and absence of hyperthermia compared with
interaction of nanotheranostics with RBCs did not induce heme- free Dox. Supporting Information Fig. S4a shows the decrease in
aggregation, which can alter the blood viscosity (Fig. S3b and survival percentage with increasing concentrations of all the
S3c). Therefore, developed nanotheranostics exhibited hemo- nanostructure and free Dox. However, MN-treated clusters
compatible behavior. showed better survival percentage than DFIO, which may be
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1717

Figure 3 (a) Hysteresis loop showing superparamagnetic properties of magnetic nanomicelles; (b) Concentration-dependent increase in SAR
values of MN; (c and d) Cumulative drug release on varying pH and alternating temperature conditions, respectively; Concentration-dependent (e)
NIR and (f) MRI imaging properties of the MN at various iron concentrations show dual diagnostic potentials.

attributed to the non-cytotoxic property of the nanocarrier. Similar apoptotic bodies after 24 h, however, PI could not penetrate due to
increase in survival percentage was observed in PEG-MN where intact cell membrane of cells in CTCs (Fig. 5). Treatment with
PEG chains inhibited diffusion of Dox from nanomicelles. CD44-MN showed an increased number of dead cells. However,
Further, a significant decrease in survival percentage was observed in the sectional view, cells in the core region of CTCs showed
in CD44-MN-treated CTCs due to rapid receptor-mediated inter- green fluorescence. Interestingly, hyperthermia treatment confirms
nalization. A significant decrease in IC50 values was observed on the occurrence of cell death throughout the cluster by the presence
subjecting clusters to hyperthermia (4.36 mg/mL) when compared of red stained cells from periphery to the core region of CTC.
to the absence of hyperthermia (6.10 mg/mL) (Table 2). Therefore, This can be due to the alternating magnetic field-induced deep
CD44-MN showed an effective decline in survival percentage in permeability of the MN into the clusters that can trigger rapid cell
the presence of hyperthermia. Hence, further experiments were death. Hence, adjuvant therapy combined with chemotherapy
carried to confirm the efficacy of cell death in CTCs. enhances rapid Dox release and kills the cells throughout the
clusters. Rapid cluster-specific internalization with complete
3.8.3. Apoptotic bodies and cell death destruction of cells in CTC would prevent any secondary tumor
CTCs were stained with acridine orange and propidium iodide to formation at distant organs.
evaluate the apoptotic bodies and cell death after treatment with
free Dox, CD44-MN in the presence and absence of hyperther- 3.8.4. Mitochondrial membrane potential
mia. Acridine orange stains the apoptotic bodies green, and pro- Depolarization of mitochondrial membrane potential is one of the
pidium iodide (PI, red) penetrated through the cellular membrane early apoptotic features of cell death and was evaluated using JC-1
in dead cells alone. CTCs treated with free Dox showed large dye (Fig. 6a). The dye in aggregated form shows integrated
1718 Ramya Dhandapani et al.

Figure 4 (a) Heterogenous CTCs treated with CD44-conjugated magnetic nanomicelles showed higher internalization than integrin (Blue:
nuclei stain, Hoechst; Red: Dox) and (b) significant decrease in cellular uptake when the CD44 receptors were blocked in CTCs (*P < 0.05,
n Z 3); (c) Prussian blue-stained CTCs treated with CD44-MN.

mitochondrial membrane (red) and on destabilization of mem- CD44-MN in the presence and absence of hyperthermia. The
brane potential, it occurs in monomeric form staining green. CTCs presence of iron ions from FIO can lead to destabilization and
treated with CD44-MN showed higher destabilization of mito- induce reactive oxygen species (ROS) that can further disintegrate
chondrial membrane potential when compared to free Dox. The cytoskeleton and DNA.
intensity of the green/red ratio was plotted (Fig. 6b) and no sig-
nificant difference in the ratios were observed after treatment with
3.8.5. Invasion studies
Table 2 IC50 estimated for different test groups. Invasion and colonization are important properties of CTCs,
Group IC50 (mg/mL) which travel from systemic circulation and develop secondary
tumors at distant organs. Treatment with CD44-MN inhibited a
Free Dox 6.58  0.8
MN 7.07  1.2 significant number of invading cells from CTCs in Boyden
CD44-MN 6.10  0.6 chamber (Fig. S4b). In the absence of targeting ligand, large
CD44-MN þ Hyp 4.36  1.5 number of invading cells were located which were, however,
DFIO 6.97  2.3 significantly lower than the free dox-treated CTCs. Fig. 6c
PEG-MN 7.18  3.1 shows a comparable number of invading cells in the presence
and absence of hyperthermia which confirms the targeted
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1719

cytotoxic potential of the nanotheranostic particles against CTCs free Dox. Free Dox showed intact tubulin (green) and DNA (blue) of
(P < 0.05). CTCs. However, CD44-MN þ hyperthermia showed complete
disruption in tubulin with no definite nucleoli/nucleus (Fig. 7a).
3.8.6. Cytoskeleton disruption and ROS generation ROS generated in the presence of hyperthermia was significantly
CTCs treated with CD44-MN in the presence and absence of hy- higher than MN and free Dox-treated CTCs. ROS generation may be
perthermia showed higher tubulin disintegration when compared to attributed to the presence of Fe3þ/2þ ions from FIO and Dox. The

Figure 5 DNA apoptosis and fragmentation of clusters post treatment in 2D, 3D and sectional view.

Figure 6 (a and b) Large fraction of JC1 monomers in hyperthermia-treated CTCs shows destabilization of mitochondrial membrane potential
(*P < 0.05, one-way ANOVA), (c) Invasion assay revealed significantly lower percentage of invaded cells after treatment in the presence of
targeted MN and hyperthermia (*P < 0.05, one-way ANOVA).
1720 Ramya Dhandapani et al.

increase in ROS was observed with decreasing GSH levels as shown 3.9. Therapeutic efficacy of nanotheranostics in immunocompetent
in Fig. 7b and c. This can be correlated to the imbalance in anti- CTC model in mice
oxidant enzymes in CTCs, leading to lipid peroxidation and sus-
ceptible to further oxidation by free radicals generated by Fenton
3.9.1. Biodistribution of nanotheranostics
reaction. Therefore, the presence of iron oxide nanoparticles can
Biodistribution studies were performed by injecting nanoparticles
stimulate Fenton reactions in cancer cells which triggers ROS
(free Dox and ICG equivalent) formulated in saline and anesthetized
generation and subsequent cellular deterioration.
before bioimaging using Invivo Xtreme, Bruker. Free Dox was
injected in the animal and observed for its preferential localization
3.8.7. Anti-colonization efficacy in heart based on its autofluorescence characteristics (Fig. 9).
The ability of the CTCs after treatment to form secondary tumors was However, untargeted MN and CD44-MN were located by the
evaluated by providing cell-adhesive platform to the clusters. Dead presence of NIR fluorescence in lungs up to 2 h, and elimination
cells were stained with sytox blue and calcein-AM was used to stain from the animal through kidneys was observed. Untargeted MN
the live cells. The clusters were treated with free Dox, MN and CD44- treatment group showed localization in lungs even after 2 h; how-
MN in the presence and absence of hyperthermia. Free Dox-treated ever, CD44-MN was located in the lungs for up to 30 min and later
groups showed higher aggressive and migrating cells with large area eliminated from kidneys. This shows minimal non-specific locali-
covered with cells where the core region of clusters showed live cells zation and reduced cardiac toxicity of targeted nanotheranostic
(Fig. 8a). This enhances the colonization potentials of CTCs at distant particles when compared to free Dox.
sites and similar results were observed in untargeted groups.
Combinatorial targeted treatment (chemotherapy þ hyperthermia) 3.9.2. In vivo CTC model in immunocompetent BALB/c mice
showed significantly reduced tumor cells migrating from clusters and Since there are no in vitro and in vivo specific CTC models
more dead cells were observed including the core region (Fig. 8b). available, in vivo CTC model was developed by intravenous
This confirms the efficacy of developed nanotheranostic particles administration of the heterogenous in vitro CTC clusters and the
against CTCs to penetrate deeper and kill them before they are tumor nodule formation was characterized in the vital organs. The
capable of forming secondary tumors. heterogenous clusters in saline were administered intravenously

Figure 7 (a) Tubulin disintegration; (b) ROS generation and (c) GSH secretion (*P < 0.05, one-way ANOVA).
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1721

Figure 8 (a) Anti-colonization assay showing CTCs in cell-adhesive platform treated with various groups; (b) area of spreading quantified with
respect to the initial size of CTCs before and after transferring (incubated for 24 h) to the cell adhesive platform (*P < 0.05, one-way ANOVA).

through tail vein of animals (n Z 4/group) with two different with saline-injected animals for 20 days. Apart from normal ani-
densities (low - 30 clusters and high - 90 clusters) to develop a mals, a similar pattern in body weight loss (Day 8) was observed
model for CTC in vivo. Bodyweight and any significant behavioral in all the animal groups including disease control animals. This
changes were recorded for 18 days. There were no significant weight loss can be correlated to the alterations in the metabolism
changes in the bodyweight pattern with low-density clusters; due to colonisation of injected CTCs and progression of disease.
however, a haphazard pattern in bodyweight and death of an an- Interestingly, targeted groups showed significant increase in the
imal (1 out of 4) was observed in animals injected with high- animal weight on subsequent treatments. Disease control and free
density clusters (Supporting Information Fig. S5a). After 18 Dox-treated groups showed steady decline in weight after Day 12
days, animals were sacrificed and lungs were stained with Bouins which can be correlated to metastatic breast cancer conditions.
stain to observe tumor nodes in the lungs (Fig. S5b). Blood There was a significant decline in the bodyweight of the diseased
collected from animals were processed to isolate circulating tumor animals (Supporting Information Fig. S6a). After 20 days, animals
cells using negative selection method and stained with CD44 to were sacrificed to collect blood and vital organs to observe any
confirm its cancer stem-cell like properties. Blood collected from secondary metastatic nodes. Weight of vital organ did not show
both low and high-density animals showed existence of tumor any significant loss between normal and diseased animals
cells in systemic circulation. However, high-density animals (Fig. S6c). Blood collected from diseased animals were cultured
showed haphazard bodyweight change and an animal death when in vitro in the presence of 6-thioguanine. Since 4T1 cells are
compared to low-density animals. Therefore, low-density CTCs resistant towards 4-thioguanine, other blood cells do not prolif-
were used for carrying out therapeutic study (Fig. S5c). Further, erate in culture dishes. Blood from diseased animals showed
animals were injected with low-density of clusters and compared nearly 89  4% area of colonies compared to normal animals
1722 Ramya Dhandapani et al.

Figure 9 Biodistribution of targeted and non-targeted nanotheranostics (tracked using NIR fluorescence) compared with distribution of free
Dox.

(Fig. S6b). Clusters also formed secondary tumors in lungs, when with and without hyperthermia groups (Fig. 10b). After second
stained with Bouins stain and showed a large number of tumor dose of treatment, further decline in CTCs would preferentially
nodes (65  5 nodes per animal) (Fig. S6b). reduce the colonization of clusters at distant organs. After Day
20, animals were sacrificed, vital organs and blood were
3.9.3. Therapeutic efficacy collected (Fig. S6e).
Therapeutic efficacy of the developed nanotheranostics can be
validated by its targeting efficacy in eliminating CTCs from 3.9.5. Blood clonogenic assay
systemic circulation and by preventing secondary tumor for- Blood cells were isolated and processed by differential centrifuga-
mation24. Therefore, the study was planned to demonstrate the tion technique to isolate tumor cells in circulation. 4T1 cells are
specific targeting and killing of CTCs before colonization by resistant towards 4-thioguanine and form colonies when cultured
providing dual therapy (chemo and adjuvant hyperthermia). The under sterile conditions in vitro, whereas other blood cells collapse
animals were divided into six groups (n Z 6) and injected with due to the toxicity of 6-thioguanine. Fig. 10c shows crystal-violet-
low density CTCs to groups 2 to 6 animals followed by treat- stained colonies after 7 days of culture and no significant differ-
ment as shown in Table 3. The dosage was given once a week ence in the number of colonies was observed between free dox,
followed with hyperthermia treatment (480 kHz for 20 min) disease control and untargeted groups (Fig. 10d). Animals treated
twice a week for Dox, MN, CD44-MN and CD44-MN þ Hyp with targeted and hyperthermia groups showed significantly
groups. The animals were observed for any behavioral changes decreased colonies (P < 0.05). This showed the effect of targeted
and bodyweight till the end of study (20 days). elimination of CTCs to prevent metastases.

3.9.4. Physiological evaluation 3.9.6. Tumor nodes in lungs

Fig. 10a shows the pictorial representation of mice subjected to Lungs were perfused with Bouins stain to visualize the tumor nodes
alternating magnetic field as adjuvant therapy after administra- formed by the circulating tumor clusters. Nodes were counted
tion of targeted nanotheranostics. Apart from normal animals, a manually and showed significant reduction in animal treated with
similar pattern in body weight loss (Day 8) was observed in all targeted particles when compared to free dox (Fig. 11a and b). In
the animal groups including disease control animals. This weight combination with hyperthermia, negligible nodes were observed in
loss can be correlated to the alterations in the body metabolism the lungs which proves the effect of dual treatment that can induce
due to colonization of injected CTCs and progression of disease spontaneous cell death of CTCs in circulation thereby preventing
in animals. Disease control and free Dox-treated groups showed further colonization. This confirms the efficacy of targeted nano-
steady decline in weight after Day 12 whereas targeted group theranostic particles to prevent metastasis in breast cancer.
showed significant increase in the animal weight on subsequent
treatments (Fig. S6d). Blood collected from animals after 7 days, 3.9.7. Histopathological evaluation
to evaluate the number of tumor cells in circulation, showed a Normal lung sections show distinct pulmonary alveoli and bron-
significant decrease when animals were treated with targeted chioles with alveolar sacs (Fig. 12a). Large obvious disseminated
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1723

Prussian blue-stained organ sections of targeted þ hyperthermia

Table 3 Group details for animal study.
groups revealed iron accumulation within cells, which can be
Group Description Treatment correlated to the effective hyperthermia treatment for inducing
1 Normal e apoptosis in tumor cells even after residing at secondary organs
2 Disease control Saline (Supporting Information Fig. S8). No predominant localization of
3 Doxorubicin (free Dox in saline) Free drug the nanoparticles in any of the vital organs shows the biocompati-
4 Untargeted (MN) Without hyperthermia bility of developed targeted magnetic nanotheranostics.
5 Targeted (CD44-MN) Without hyperthermia
6 Targeted (CD44-MN) With Hyperthermia
4. Conclusions

multiple secondary metastases with adjacent destruction and Targeted magnetic nanotheranostics were engineered using
boundary loss of lung were observed in disease control group ani- amphiphilic chitosan and superparamagnetic iron oxide nano-
mals treated with saline. Similar metastatic nodules were identified particles exhibiting multi-modal, multi-responsive drug release and
in free Dox and untargeted groups confirming progressive sec- combinatorial treatment against elimination of CD44-expressing
ondary tumor formation at distant organs in the presence of circulating tumor clusters. Results confirmed excellent hemo- and
chemotherapy and non-specific nanotheranostic particle treatment. cytocompatibility of nanotheranostics with specific CD44-mediated
However, treatment with the targeted nanotheranostics in the internalization in CTCs. The developed nanotheranostic particles
presence and absence of hyperthermia showed no obvious exhibited concentration dependent NIR and MRI dual diagnostic
disseminated multiple secondary metastases and significantly potentials facilitating tracking of small clusters and their coloniza-
decreased number of tumor nodules (Fig. 12b). Interestingly, tion at distant organs. The synergistic cytotoxicity contributed by
similar results were observed in liver sections where a significant Dox released from nanotheranostics and magnetic hyperthermia
increase in the sporadic and small metastases in animals without showed spontaneous cell death and anti-colonization potentials
hyperthermia treatment was observed (Fig. 12c and d). This con- against CTCs. Interestingly, magnetic nanomicelles specifically
firms the efficacy of targeted therapy, along with adjuvant ther- targeted and eliminated the colonization of heterogenous CTCs
motherapy, which showed a reduced number of secondary injected in vivo in lungs and liver when compared to disease control
metastases in lung and liver. Histopathological evaluation showed animals. Also, reduced number of tumor cells in circulation were
no significant damage to the native tissue microarchitecture and evident through colony forming assay. There was no significant
functionality in other vital organs (Supporting Information Fig. S7). accumulation of iron particles in the vital organs, confirming the

Figure 10 (a) Pictorial representation of mice under hyperthermia; (b) Tumor cells isolated from blood at Day 7 of administration; (c) Colonies
of tumor cells from blood collected from animals at Day 20; (d) Graphical representation of percentage of colonies formed in each group
(*P < 0.05, n Z 4).
1724 Ramya Dhandapani et al.

Figure 11 (a) Tumor nodes in Bouins stained lungs visualized under stereomicroscope; (b) Graphical representation of the number of tumor
nodes in lungs (*P < 0.05).

Figure 12 (a and b) H& E staining of lung shows predominant tumor nodules in disease control group, free Dox-treated and untargeted groups.
Targeted in the presence and absence of hyperthermia shows lung histology similar to normal group with no significant secondary nodes as per
histopathological scoring shown in box plot. (c and d) Liver sections showed small metastases in disease control, free Dox and untargeted groups
whereas hyperthermia and targeted groups confirmed equivalent histology to that of normal group (one-way ANOVA) (Black arrows: granulocyte
infiltrates; Red arrow: sporadic and small metastases).
CD44 expressing circulating tumor clusters targeted multifunctional nanotheranostic systems 1725

biocompatible nature of developed magnetic nanomicelles. Hence, 8. Riebensahm C, Joosse SA, Mohme M, Hanssen A, Matschke J, Goy Y,
targeted nanotheranostics engineered against heterogenous CTCs et al. Clonality of circulating tumor cells in breast cancer brain
can successfully eliminate colonization and secondary tumor for- metastasis patients. Breast Cancer Res 2019;21:101.
mation, which would be a potential theranostic approaches to in- 9. Yan W, Lang T, Zhu R, Zhu X, Li Y, Wu T, et al. Anti-hypoxia
nanosized drug delivery systems improving cancer therapy. Nano
crease the survival rate of the metastatic patients.
Today 2022;42:101376.
10. Leary M, Heerboth S, Lapinska K, Sarkar S. Sensitization of drug
Acknowledgments resistant cancer cells: a matter of combination therapy. Cancers
(Basel) 2018;10:483.
Authors thank Nano Mission (SR/NM/NS-1205/2015(G)), PG- 11. Li D, Wang Y, Li C, Wang Q, Sun B, Zhang H, et al. Cancer-specific
Teaching (SR/NM/PG-04/2015), and FIST (SR/FST/LSI-622/ calcium nanoregulator suppressing the generation and circulation of
2014), Department of Science and Technology, Government of circulating tumor cell clusters for enhanced anti-metastasis combina-
India for financial support. Ramya Dhandapani is thankful to tional chemotherapy. Acta Pharm Sin B 2021;11:3262e71.
12. Huang X, Brazel CS. On the importance and mechanisms of burst
Council of Scientific and Industrial Research for senior research
release in matrix-controlled drug delivery systems. J Control Release
fellowship (09/1095(0022)/18-EMR-I), Government of India.
2001;73:121e36.
13. Dhandapani R, Subramanian A, Sethuraman S. ECM-mimetic
Author contributions multiresponsive nanobullets targeted against metastasizing circu-
lating tumor clusters in breast cancer. Ann Biomed Eng 2020;48:
Ramya Dhandapani performed experiments, analyzed data and 568e81.
wrote the manuscript. Uma Maheswari Krishnan and Swaminathan 14. Beltrán-Gracia E, López-Camacho A, Higuera-Ciapara I, Velázquez-
Sethuraman reviewed the manuscript. Anuradha Subramanian Fernández JB, Vallejo-Cardona AA. Nanomedicine review: clinical
conceived the project and designed experiments. All authors dis- developments in liposomal applications. Cancer Nano 2019;10:11.
15. Trivedi R, Kompella UB. Nanomicellar formulations for sustained
cussed the results and reviewed the manuscript.
drug delivery: strategies and underlying principles. Nanomedicine
(Lond) 2010;5:485e505.
Conflicts of interest 16. Lu Y, Yue Z, Xie J, Wang W, Zhu H, Zhang E, et al. Micelles with
ultralow critical micelle concentration as carriers for drug delivery.
Authors have no conflicts of interest. Nat Biomed Eng 2018;2:318e25.
17. Sharma D, Singh J. Synthesis and characterization of fatty acid grafted
Appendix A. Supporting information chitosan polymer and their nanomicelles for nonviral gene delivery
applications. Bioconjug Chem 2017;28:2772e83.
18. Chen H, Ye Z, Sun L, Li X, Shi S, Hu J, et al. Synthesis of chitosan-
Supporting data to this article can be found online at https://doi. based micelles for pH responsive drug release and antibacterial
org/10.1016/j.apsb.2022.12.003. application. Carbohydr Polym 2018;189:65e71.
19. Manigandan A, Handi V, Sundaramoorthy NS, Dhandapani R,
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