CD44 Targeted Nanomaterials for Treatment of Triple-Negative Breast Cancer

Simple Summary Triple-negative breast cancer (TNBC) is one of the most challenging tumors with aggressive behavior, low recovery rate, poor prognosis, high metastatic potential, and rapid relapse compared to other breast cancer subtypes. Conventional therapies currently have minimal effect on TNBC; thus, using combination therapies is a valid strategy to enhance drug activity and minimize the overall adverse effect. Therefore, combining drugs with a different mechanism of actions such as apoptosis inducers and JAK/STAT3 inhibitors improved TNBC cell lines killing activity in vitro and in vivo. To further improve the hydrophobic drug activity, CD44 targeted polymeric nanoparticles (CD44-T-PNPs) were utilized by encapsulating hydrophobic drug (CFM-4.16) in CD44-T-PNPs to enhance the drug solubility, tumor accumulation, and most importantly, enhance drug potency. Tagging our PNPs with Hyaluronic acid (HA) enhanced tumor accumulation, reduced off-target distribution, and improved therapeutic efficacy. Abstract Identified as the second leading cause of cancer-related deaths among American women after lung cancer, breast cancer of all types has been the focus of numerous research studies. Even though triple-negative breast cancer (TNBC) represents 15–20% of the number of breast cancer cases worldwide, its existing therapeutic options are fairly limited. Due to the pivotal role of the presence/absence of specific receptors to luminal A, luminal B, HER-2+, and TNBC in the molecular classification of breast cancer, the lack of these receptors has accounted for the aforementioned limitation. Thereupon, in an attempt to participate in the ongoing research endeavors to overcome such a limitation, the conducted study adopts a combination strategy as a therapeutic paradigm for TNBC, which has proven notable results with respect to both: improving patient outcomes and survivability rates. The study hinges upon an investigation of a promising NPs platform for CD44 mediated theranostic that can be combined with JAK/STAT inhibitors for the treatment of TNBC. The ability of momelotinib (MMB), which is a JAK/STAT inhibitor, to sensitize the TNBC to apoptosis inducer (CFM-4.16) has been evaluated in MDA-MB-231 and MDA-MB-468. MMB + CFM-4.16 combination with a combination index (CI) ≤0.5, has been selected for in vitro and in vivo studies. MMB has been combined with CD44 directed polymeric nanoparticles (PNPs) loaded with CFM-4.16, namely CD44-T-PNPs, which selectively delivered the payload to CD44 overexpressing TNBC with a significant decrease in cell viability associated with a high dose reduction index (DRI). The mechanism underlying their synergism is based on the simultaneous downregulation of P-STAT3 and the up-regulation of CARP-1, which has induced ROS-dependent apoptosis leading to caspase 3/7 elevation, cell shrinkage, DNA damage, and suppressed migration. CD44-T-PNPs showed a remarkable cellular internalization, demonstrated by uptake of a Rhodamine B dye in vitro and S0456 (NIR dye) in vivo. S0456 was conjugated to PNPs to form CD44-T-PNPs/S0456 that simultaneously delivered CFM-4.16 and S0456 parenterally with selective tumor targeting, prolonged circulation, minimized off-target distribution.

. Momelotinib and CFM4.16 synergistic combination. Momelotinib is a JAK-STAT inhibitor. JAK-STAT pathway is a direct signaling pathway transfer the signal from extracellular to nucleus to control the expression of certain genes. The upregulation of the JAK-STAT pathway is the key player in different cancers, and its regulation is under clinical investigation for cancer therapy. The principal components of this pathway are: cytokines-receptor complex, JAK, and STAT proteins. Mechanistically, when the ligand bind to its corresponding JAK-associated receptor (1), the receptors arms are brought into proximity, which enables transphosphorylation between the two JAK molecules (2). The activated phosphorylated JAK subsequently phosphorylates the receptor arms, which is the binding site for the latent transcription factors STAT (3). After the STAT molecules bind to the receptor arms (4), they become ready for phosphorylation by JAK (5). Once phosphorylated, the two STAT monomers dimerize through reciprocal phosphotyrosine-SH2 domain interaction (6). The STAT dimer is an active transcriptional factor that is translocated to the nucleus (7) and binds to a specific DNA sequence in the target gene promoters by DNA-binding domain to control transcription of specific genes (8). Momelotinib Momelotinib is a JAK-STAT inhibitor. JAK-STAT pathway is a direct signaling pathway transfer the signal from extracellular to nucleus to control the expression of certain genes. The upregulation of the JAK-STAT pathway is the key player in different cancers, and its regulation is under clinical investigation for cancer therapy. The principal components of this pathway are: cytokines-receptor complex, JAK, and STAT proteins. Mechanistically, when the ligand bind to its corresponding JAK-associated receptor (1), the receptors arms are brought into proximity, which enables transphosphorylation between the two JAK molecules (2). The activated phosphorylated JAK subsequently phosphorylates the receptor arms, which is the binding site for the latent transcription factors STAT (3). After the STAT molecules bind to the receptor arms (4), they become ready for phosphorylation by JAK (5). Once phosphorylated, the two STAT monomers dimerize through reciprocal phosphotyrosine-SH2 domain interaction (6). The STAT dimer is an active transcriptional factor that is translocated to the nucleus (7) and binds to a specific DNA sequence in the target gene promoters by DNA-binding domain to control transcription of specific genes (8). Momelotinib antagonizes the ATP binding to JAK1/2, leading to inhibition of the JAK-STAT pathway. CFM4.16 is CARP-1/APC/C interaction inhibitor. APC/C is E3 ubiquitin ligase responsible for tagging cell cycle proteins for proteasomal degradation for the metaphase/ anaphase cell cycle transition. Aberrant APC/C system is associated with cancer progression. Mechanistically, the process starts with latent ubiquitin (Ub) molecules present in the cells (1 ), which is activated by Ub-activating enzymes (E1) in an ATPdependent manner (2 ). The activated (Ub)will be transferred to a Ub-conjugating enzyme (E2) (3 ), which will conjugate the Ub molecules to activated Ub ligase (E3)/APC/C. The E3/APC/C is under the control of CDK-1(cyclin-dependent kinase-1), CARP-1, and CDC 20 (cell division cycle protein 20).The CDK-1 phosphorylates the APC/C, while CARP-1 binds to the APC2 subunit of APC/C for coactivation. Then the phosphorylated APC/C will bind to CDC 20 to be fully activated (4 ). Then The (E2) conjugates the Ub molecules to activated Ub ligase (E3)/(APC/C) (5 ). Activated APC/C system ubiquitinates the securin protein (chaperone) to be marked for proteasomal degradation (6 &7 ). After the degradation of securin, the separase (separin) will be activated and will break down the cohesin protein between two sister chromatids (8 ). Break down of cohesin will lead to the separation of two sister chromatids in anaphase (10 ). Thus, APC/C is responsible for maintaining normal chromosome number and genetic stability. Also, APC/C is responsible for turning over S/M cyclins to terminate mitosis. The proteasome catalytic unit will degrade the tagged protein into a small peptide chain and Ub. The Ub will be reused, and the fate of the peptide chain will depend on the cell needs; either it will be repurposed for protein synthesis or energy production (9 ). The simultaneous down-regulation of STAT3 and APC/C activation is the underlying mechanism for their synergism.
Furthermore, the study has developed an apoptosis inducer (CFM-4.16) that stands for CARP-1 functional mimetics (CFMs). CARP-1/CCAR1 (Cell cycle and apoptosis regulator 1) is a peri-nuclear phospho-protein which plays a vital role in regulating cell proliferation and apoptosis pathways. Two E3 ubiquitin ligases govern the cell cycle; APC/C (anaphasepromoting complex/cyclosome) and SCF (Skp1, Cullins, F-box proteins). They tag various regulatory proteins with ubiquitin to be degraded with the proteasome to control many cell functions such as cell cycle, signal transduction, and DNA replication [14,15]. APC/C mediates metaphase/anaphase cell cycle transition and it is under the control of CDK-1(cyclin-dependent kinase-1), CARP-1, and CDC 20 (cell division cycle protein 20). CFM 4.16 is inhibitor of CARP-1-APC/C interaction in APC2 subunit leading to (1) interfere with the cell cycle transition function of APC/C; (2) accumulation of CARP-1. Accumulated CARP-1 induces apoptosis by stimulating tumor suppressors, such as p53, caspase-9, and p38 MAPK, and inhibiting oncogenes, such as c-Met and c-Myc. CARP-1 knocking -down resulted in apoptosis resistance, which indicates the importance of CARP-1 for cell proliferation and apoptosis [16,17], as shown in Figure 1.
Following the recommendation to apply a simultaneous combination therapy in cases of urgent mitigation of tumor burden is required, especially in advanced metastatic cancers, the study has opted for a co-administration MMB and CFM-4.16 for malignant TNBC. In an attempt to surpass the outcome of monotherapy, the employed simultaneous combination paradigm has pivoted upon a repurposing of combined old conventional drugs-reaching the result that: Simultaneous combination overcomes multidrug resistance with increased survivability [8].
Due to their illustrated ability to improve chemotherapy safety profile, enhance cancer targetability, allow burst/sustained drug release, and prevent premature drug degradation, nanoparticles (NPs) have been lately extensively investigated-particularly their effectiveness for cancer therapy [18]. One NP cargo could be used for theranostic purpose or a simultaneous combination. The bioavailability of NPs-encapsulated drugs can be improved by anti-fouling or zwitterionic agents, as they will delay NP renal filtration and prevent non-specific accumulation in the reticuloendothelial system (RES) [18]. Many nanoformulations are successfully marketed for cancer therapy as they can overcome the limitation of conventional anticancer therapy such as Doxil ® , Abraxane ® , and Genexol-PM ® [18].
In the study, the employed polymeric NPs (PNPs)-one of multiple NPs platforms which consists of block copolymers, D-alpha-tocopheryl polyethylene glycol succinate (Vitamin E TPGS), and styrene-maleic acid (SMA)-showed improvement in drug solubility and in vitro and in vivo biodistribution. The biocompatibility and degradability of the chosen nanoplatform made it the right candidate for hydrophobic drug delivery. TPGS is an approved FDA delivery carrier due to its inherent preferential features, as well as its ability to inhibit P-glycoprotein (P-gp) associated with multidrug resistance (MDR) [19]. SMA is well-suited for clinical translation due to low cost and ease of processing [20,21]. PNPs surface can be decorated with targeting molecules to increase the bio-affinity to cancerous cells. Hyaluronic acid (HA) is the targeting ligand for a cluster of differentiation-44 (CD44) receptors. CD44 normally expressed in embryonic cells, bone marrow, and connective tissue, CD44 is abnormally extensively expressed in pancreatic, breast, and lung cancers, especially in stem cell subpopulations. CD44 expression indicates poor prognosis, metastasis, EMT mediated chemotherapeutic resistance, and low survivability. For that matter, many contemporary studies support the potential benefits of combining chemo or radiotherapy with CSCs-targeting therapy to overcome tumor resistance and relapse. CD44 in breast cancer is associated with increased (P-gp) and Bcl gene expression responsible for MDR and apoptosis resistance. Based on the clinicopathological impact of CD44 in breast cancer basal type, it is applied as a molecular diagnostic marker, prognostic tool, therapeutic target, targeting ligand-receptor in various stages of clinical development. It is worth noting that whereas CD44 binds to several ligands such as chondroitin, osteopontin, fibronectin, collagen, and serglycin/sulfated proteoglycan, HA remains the specific ligands for CD44 and its all isomers. Moreover, HA is considered the main extracellular matrix component expressed by cancer and stromal cells [22]. HA is widely implemented in cancer therapies due to its intrinisic properties such as biocompatability, biodegradability, safety, non-immungenicity, non-inflammatory, anionic charge, simple linear structure, and ease processing by modifing its funcional groups such as carboxy, hydroxy and N-acetyl groups. Based on the above, tagging our PNPs with HA enhanced tumor accumulation, reduced off-target distribution, and improved therapeutic efficacy.

The Cytotoxicity of The Individual Drugs and Their Combinations Studies
Both MMB and CFM-4.16 exhibited dose-dependent cytotoxicity with IC50 values of (4.2 & 3.4 µM) and (10.8 & 12.8 µM) in MDA-MB-231 and MDA-MB-468, respectively at 72 h as illustrated in Figure 2A,B. MMB potentiated the cytotoxicity of CFM-4.16 in TNBC cell lines with marked dose reduction and cell whipping, as illustrated in Figure 2 and Figure S1, at which the data is analyzed by COMPUSYN software. The isobolograms in Figure 2C showed that MMB + CFM-4.16 combination had many synergistic points with CI < 1, while others with CI>1 are antagonistic and those with CI = 1 are additive. The MMB + CFM-4.16 showed effective synergism accompanied by high fraction affected (Fa > 0.5) and CI < 1, as shown in Figure 2D. Also, MMB reduces the required concentration of CFM-4.16, as it is evident by (DRI) in Figure 2E. Combination points with DRI > 1 is favorable, DRI = 1 are with no effect, and DRI < 1 are unfavorable. All the CI points of the MMB + CFM-4.16 in both MDA-MB-231 and MDA-MB-468 are present in the Figure S1. MMB + CFM-4. 16 Targeted SMA-TPGS Carrier and Targeted  HA-SMA-TPGS-Carrier The synthesized non-targeted SMA-TPGS (NT-PNPs) and targeted HA-SMA-TPGS (CD44-T-PNPs) carriers were characterized by FTIR and 1 H NMR spectroscopy, as shown in (Figures S2 and S3). The reaction between TPGS and SMA generates SMA-TPGS compound, which was confirmed in IR spectra by the presence of four characteristic bands at 3500, 3000,1750, 1250 cm −1 due to O-H, C-H of aromatic hydrocarbon, C=O, and C-O-C groups, respectively. 1 H NMR spectrum of SMA-TPGS shows the characteristic chemical shift at 6.685-7.153 ppm (aromatic H peaks of SMA) and 4.014 ppm (CH2-O-of TPGS). While HA, TPGS, and SMA underwent three pots reaction to produce a product identified as HA-SMA-TPGS. The IR spectra exhibit four bands at 3500, 3100, 1750, 1150, and 1050 cm −1 due to OH, C-H of aromatic hydrocarbon, C=O, C-N, and C-O-C groups, respectively. N-H band is overlapped with the O-H band in the IR spectrum. 1 H NMR spectrum of HA-SMA-TPGS shows the characteristic chemical shift at 6.711-7.2 ppm (aromatic H peaks of SMA), 4.014 ppm (CH2-O-of TPGS), and 4.4-4.6 ppm (hyaluronic acid H in sugar rings). The retention of the characteristic IR and 1 H NMR peaks of the individual monomers (HA, SMA, TPGS) in the produced SMA-TPGS and HA-SMA-TPGS indicate successful coupling and confirm the formation of the conjugated polymers. The nontargeted and targeted conjugates will be self-assembled into PNPs in the aqueous media due to the presence of hydrophobic SMA polymer and hydrophilic TPGS and HA polymers. The formed PNPs will be water-soluble with a hydrophobic core. The hydrophobic core could be physically or chemically incorporated with hydrophobic drugs for parenteral administration.

Preparation and Characterization of CFM-4.16 Loaded Polymeric Nanoparticles (PNPs)
The TEM revealed that NT-PNPs and CD44-T-PNPs are spherical with a smooth surface. The DLS average particle size of NT-PNPs was 81.5 nm and of CD44-T-PNPs was 98.1 nm with a narrow polydispersity index (PDI) 0.176 and 0.169, respectively, consistent with TEM results. The surface charge of NT-PNPs was 6.57 ± 2.94 mV and of CD44-T-PNPs was -7.25 ± 2.94, as shown in (Figure 3). CD44-T-PNPs had a larger particle size and negative zeta potential, attributed to wrapping the PNPs surface with HA. The water solubility of both formulations favors their parenteral administration. The loading contents (LC%), encapsulation efficiency (EE%), and yield% of NT-PNPs and CD44-T-PNPs are presented in (Table 1) as mean ± SD, n = 3. The synthesized non-targeted SMA-TPGS (NT-PNPs) and targeted HA-SMA-TPGS (CD44-T-PNPs) carriers were characterized by FTIR and 1 H NMR spectroscopy, as shown in (Figures S2 and S3). The reaction between TPGS and SMA generates SMA-TPGS compound, which was confirmed in IR spectra by the presence of four characteristic bands at 3500, 3000,1750, 1250 cm −1 due to O-H, C-H of aromatic hydrocarbon, C=O, and C-O-C groups, respectively. 1  The nontargeted and targeted conjugates will be self-assembled into PNPs in the aqueous media due to the presence of hydrophobic SMA polymer and hydrophilic TPGS and HA polymers. The formed PNPs will be water-soluble with a hydrophobic core. The hydrophobic core could be physically or chemically incorporated with hydrophobic drugs for parenteral administration.

Preparation and Characterization of CFM-4.16 Loaded Polymeric Nanoparticles (PNPs)
The TEM revealed that NT-PNPs and CD44-T-PNPs are spherical with a smooth surface. The DLS average particle size of NT-PNPs was 81.5 nm and of CD44-T-PNPs was 98.1 nm with a narrow polydispersity index (PDI) 0.176 and 0.169, respectively, consistent with TEM results. The surface charge of NT-PNPs was 6.57 ± 2.94 mV and of CD44-T-PNPs was -7.25 ± 2.94, as shown in (Figure 3). CD44-T-PNPs had a larger particle size and negative zeta potential, attributed to wrapping the PNPs surface with HA. The water solubility of both formulations favors their parenteral administration. The loading contents (LC %), encapsulation efficiency (EE %), and yield % of NT-PNPs and CD44-T-PNPs are presented in (Table 1) as mean ± SD, n = 3.     Figure 4B. Higher cellular uptake of CD44-T-PNPs affirmed in both cell lines is probably due to receptor-mediated endocytosis followed by HA/CD44 interaction. CD44-targeted nanomaterials could be a useful tool for selective cytotoxicity in TNBC.   Figure 4. Blue fluorescence refers to Hoechst stained nuclei, while red fluorescence refers to the Rhodamine B uptake signal. In MDA-MB-231, CD44-T-PNPs/Rhod had 2.5 folds better tumor accumulation compared to NT-PNPs/Rhod, as shown in Figure 4A. While in MDA-MB-468, the CD44-T-PNPs had 2.1 folds better tumor accumulation compared to NT-PNPs, as shown in Figure 4B. Higher cellular uptake of CD44-T-PNPs affirmed in both cell lines is probably due to receptor-mediated endocytosis followed by HA/CD44 interaction. CD44-targeted nanomaterials could be a useful tool for selective cytotoxicity in TNBC.

Targeted PNPs Combination has Exhibited Remarkable Anticancer Activity Compared to Free Drugs Against TNBC Cell Lines
The MMB+CFM-4.16 combination had a more cytotoxic effect than individual MMB and CFM-4.16, as confirmed Figure Figure 6. Interestingly, T combo (CD44-T-PNPs +MMB) had a significant potent cytotoxic effect compared to the free combo, as shown in Figure 6. T combo decreased the cells viability percentage by 2.5 and 2.16 folds in MDA-MB-231 and MDA-MB-461, respectively, compared to the free combo (MMB+CFM-4.16). Moreover, the results illustrated in Figure 7 show a decrease in the proliferation capacity, increase in the cellular detachment, and significant change in the cellular morphology in the combination wells compared to control negative. In combinations wells, especially the targeted one, the cells appear as a star shape with tapered ends with very low density. The co-treatment of MMB + CFM4.16 either in free or in PNPs form inhibited cell migration and wound closure at both 24 and 72 h.   Figure 7 show a decrease in the proliferation capacity, increase in the cellular detachment, and significant change in the cellular morphology in the combination wells compared to control negative. In combinations wells, especially the targeted one, the cells appear as a star shape with tapered ends with very low density. The co-treatment of MMB + CFM4.16 either in free or in PNPs form inhibited cell migration and wound closure at both 24 and 72 h.      The ability of our combinations to induce ROS-dependent apoptosis is shown in Figure 8A,B. Individually, drugs exhibited a slight increase in ROS production in both MDA-MB-231 and MDA-MB-468. In MDA-MB-231, the ROS generation was increased by 1. 55 16, free combo, NT combo, and T combo, respectively compared to negative control. The NT and T carriers had a slight attenuation in ROS production due to vitamin E's antioxidant effect in their content [23]. These results suggested that MMB + CFM-4.16 synergistic effect due to the increase in ROS generation. Moreover, T combo, with its unprecedented ROS production, alter the redox environment promoting oxidative stress-induced cancer cell death.  16, free combo, NT combo, and T combo, respectively compared to negative control. The NT and T carriers had a slight attenuation in ROS production due to vitamin E's antioxidant effect in their content [23]. These results suggested that MMB + CFM-4.16 synergistic effect due to the increase in ROS generation. Moreover, T combo, with its unprecedented ROS production, alter the redox environment promoting oxidative stress-induced cancer cell death. In addition, caspase 3 and caspase 7 which are crucial for the execution phase of cellular apoptosis have been also investigated. Caspase 3 activation is a conclusive marker for the irreversible commitment of cellular apoptosis. The results have revealed enhancement of caspase 3/7 activity in the free combo compared to the individual drugs and control groups in both cell lines. In MDA-MB-231, caspase activity increased by 1.13 for MMB, 1.39 for CFM-4.16, 1.52 for the free combo, and 1.62 for NT combo compared to negative control cells. In MDA-MB-468, caspase activity increased by 1.18 for MMB, 1.25 for CFM-4.16, 1.67 for the free combo, and 2.01 for NT combo compared to negative control cells. Interestingly, the T combo markedly stimulated caspase 3/7 activity over the free combo; by 1.76 in MDA-MB-231 and 2.14 folds MDA-MB-468, as shown in Figure 9. In addition, caspase 3 and caspase 7 which are crucial for the execution phase of cellular apoptosis have been also investigated. Caspase 3 activation is a conclusive marker for the irreversible commitment of cellular apoptosis. The results have revealed enhancement of caspase 3/7 activity in the free combo compared to the individual drugs and control groups in both cell lines. In MDA-MB-231, caspase activity increased by 1.13 for MMB, 1.39 for CFM-4.16, 1.52 for the free combo, and 1.62 for NT combo compared to negative control cells. In MDA-MB-468, caspase activity increased by 1.18 for MMB, 1.25 for CFM-4.16, 1.67 for the free combo, and 2.01 for NT combo compared to negative control cells. Interestingly, the T combo markedly stimulated caspase 3/7 activity over the free combo; by 1.76 in MDA-MB-231 and 2.14 folds MDA-MB-468, as shown in Figure 9.   The expression of CD44 in ectopic tumor xenograft collected from the TNBC-bearing mice model was investigated by immunohistochemistry. The intense bright green fluorescence indicates the high expression level of CD44, as shown in Figure 11. Molecular characterization leads to the discovery of biomarkers and targeted therapy, which is the basis of personalized medicine. The association of CD44 with tumorigenesis induction, poor prognosis, aggressiveness, relapse, and chemotherapeutic resistance of TNBC has been the underlying rationale behind the study's choice of CD44 as an excellent biomarker for site-specific payload delivery to TNBC.

Animal Studies 2.4.1. CD44 Receptors Are Overexpressed in Tumors of TNBC-Bearing Mice Model
The expression of CD44 in ectopic tumor xenograft collected from the TNBC-bearing mice model was investigated by immunohistochemistry. The intense bright green fluorescence indicates the high expression level of CD44, as shown in Figure 11. Molecular characterization leads to the discovery of biomarkers and targeted therapy, which is the basis of personalized medicine. The association of CD44 with tumorigenesis induction, poor prognosis, aggressiveness, relapse, and chemotherapeutic resistance of TNBC has been the underlying rationale behind the study's choice of CD44 as an excellent biomarker for site-specific payload delivery to TNBC.

NIR Imaging and Biodistribution and Inducible DNA-DSBs
The theranostic PNPs is an emerging aspect of a precise medicine. It consists of targeting ligand, therapeutic agents, and imaging agents. Sufficiently accumulated in the tumor by enhanced permeability and retention (EPR) and receptor-mediated endocytosis, theranostic NPs is helpful for early diagnosis, image-guided surgery, and tracking drug distribution, accumulation, sustained release, and efficacy. Being less toxic and cost-effective, NIR imaging in TNBC-bearing mice has been opted for by the study. The results have illustrated significant tumor homing of CD44-T-PNPs/S0456, followed by NT-PNPs/S0456, compared to control (free S0456) at both 24 and 72 h, as shown in Figure 12. In addition, the CD44-T-PNPs/S0456 exhibits no off-target accumulation, especially in the liver, compared to NT-PNPs/S0456 at both 24 and 72 h as shown in whole-body and dissected organ imaging Figure 12. The high intensity of the CD44-T-PNPs/S0456 group in the kidney at 72 h has revealed the following (1) hydrophilic nature of the formulation enabled its renal clearance; (2) the formulation had prolonged sustainable property till 72 h; (3) renal mediated excretion reduces liver toxicity; (4) HA surface coating reduced NPs immunogenicity and elimination by RES. The selective homing of CD44 targeted PNPs profoundly support the rational application and clinical translation of it to the theranostic platform of metastatic TNBC. To support the therapeutic efficacy, a TUNEL assay was performed-indicating the results that: the T combo was significantly able to push the tumor cells to late-stage apoptosis when compared to individual drugs, as shown in

NIR Imaging and Biodistribution and Inducible DNA-DSBs
The theranostic PNPs is an emerging aspect of a precise medicine. It consists of targeting ligand, therapeutic agents, and imaging agents. Sufficiently accumulated in the tumor by enhanced permeability and retention (EPR) and receptor-mediated endocytosis, theranostic NPs is helpful for early diagnosis, image-guided surgery, and tracking drug distribution, accumulation, sustained release, and efficacy. Being less toxic and costeffective, NIR imaging in TNBC-bearing mice has been opted for by the study. The results have illustrated significant tumor homing of CD44-T-PNPs/S0456, followed by NT-PNPs/S0456, compared to control (free S0456) at both 24 and 72 h, as shown in Figure 12. In addition, the CD44-T-PNPs/S0456 exhibits no off-target accumulation, especially in the liver, compared to NT-PNPs/S0456 at both 24 and 72 h as shown in whole-body and dissected organ imaging Figure 12. The high intensity of the CD44-T-PNPs/S0456 group in the kidney at 72 h has revealed the following (1) hydrophilic nature of the formulation enabled its renal clearance; (2) the formulation had prolonged sustainable property till 72 h; (3) renal mediated excretion reduces liver toxicity; (4) HA surface coating reduced NPs immunogenicity and elimination by RES. The selective homing of CD44 targeted PNPs profoundly support the rational application and clinical translation of it to the theranostic platform of metastatic TNBC. To support the therapeutic efficacy, a TUNEL assay was performed-indicating the results that: the T combo was significantly able to push the tumor cells to late-stage apoptosis when compared to individual drugs, as shown in Figure 13.

Discussion
TNBC is one of the most challenging tumors with an aggressive behavior, low recovery rate, poor prognosis, high metastatic potential, and rapid relapse compared to other breast cancer subtypes. TNBC abbreviation derived from the deprivation of three types of

Discussion
TNBC is one of the most challenging tumors with an aggressive behavior, low recovery rate, poor prognosis, high metastatic potential, and rapid relapse compared to other breast cancer subtypes. TNBC abbreviation derived from the deprivation of three types of Figure 13. The capability of T combo to enhance the onset of apoptosis in TNBC-bearing mice by CD44. HA mediated endocytosis. T combo was able to drive the tumor to the late-stage apoptosis, which is based on generating multiple DNA double-strand breaks (DSBs) with accessible 3'-hydroxyl (3'-OH) groups that were detected by TUNEL. Elevated apoptosis is indicated by brownish discoloration or dark brown spots. Note: T combo (CD44-T-PNPs + MMB) where MMB means momelotinib. Magnification is 40×.

Discussion
TNBC is one of the most challenging tumors with an aggressive behavior, low recovery rate, poor prognosis, high metastatic potential, and rapid relapse compared to other breast cancer subtypes. TNBC abbreviation derived from the deprivation of three types of receptors; estrogen receptors (ER), progesterone receptor (PR), and human epidermal growth factor (HER2) [24]. The discovery of these biomarkers by Perou 2000 played an essential role in the development of targeted, personalized medicine. Due to the lack of these receptors, TNBC patients can not benefit from currently available receptor-targeted systemic therapy, making surgery and chemotherapy the only available option.
However, traditional chemotherapeutic agents' application has not been without potential side effects, suboptimal outcomes, and tumor resistance development. In addition, even though most of the chemotherapeutic agents have the capacity to attack fast-growing cancerous cells, they wipe out fast-growing healthy cells too, such as: bone marrow cells, hair follicle cells, and cells lining the gastrointestinal tract (GIT) [25][26][27]. In the same vein, despite the fact that most chemotherapeutic agents can trim tumor growth, its effect is not long-lasting and is followed by rapid proliferation and invasion, which is the reason for the development of chemotherapeutic resistance. Drug resistance is considered the main obstacle that breast cancer patients have to confront and is responsible for chemotherapeutic failure. The hurdle of drug resistance could be innate, acquired, or cross-resistance/MDR with different underlying mechanisms such as drug sequestration by P-gp, proliferation potential of mutated cancer stem cells (CSCs), altering drug target, modification of DNA repair strategies, altering drug detoxification, and invalid apoptotic regulators such as p53 [28,29]. There upon appears the urgent need for new approaches such as immunotherapeutic approach [30], new agents that do not exhibit cross-resistance such as ixabepilone [28], targeting cancer stem cells, and unique combination strategies.
The current study establishes a novel combination strategy based on the sensitization of TNBC to CFM-4.16 by MMB, which has proven an unequivocally capacity to overcome single-agent prone -therapeutic resistance, reduce systemic toxicity, and enhance the therapeutic index. Moreover, CFM-4.16 water solubility was enhanced by SMA-TPGS carrier as well as cell uptake using CD44 targeting ligand in vitro and in vivo. It is worth noting that the possibility of using this cargo for the theranostic purpose has been likewise investigated.
PNPs were first nominated for cancer therapy in the early 1980s. PNPs able to increase the water solubility of hydrophobic drugs as they consist of a hydrophilic shell, which interacts with the external aqueous media, and a hydrophobic core, which acts as a depository for hydrophobic drugs [31]. This is in addition to their ability to enhance drug retention in tumor tissue by EPR effect as well as prolonging plasma half-lives by escaping renal elimination [18,32]. One of the polymeric micelles that have been FDA approved for breast cancer is Genexol-PM ® [33]. The study has opted for a carrier, which is a block copolymer consisting of SMA and Vitamin E-TPGS, decorated by HA as a targeting ligand. Established as biologically safe and immunostimulant [34,35] SMA was clinically approved for the treatment of hepatoma in Japan in 1993 [36,37]. Having a high glass transition temperature, it increases NPs stability and controls the drug release. Moreover, the carboxyl group of maleic acid on SMA's hydrophilic surface enables surface modification by conjugation to the targeting ligand, such as HA [20,21,38]. Vitamin E-TPGS is FDA approved drug adjuvant which is widely used as a pharmaceutical emulsifier, stabilizer, and permeation and bioavailability enhancer of hydrophobic drugs with a potent P-gp inhibition and apoptosis induction [19,39,40].
PNPs is a promising drug delivery to overcome poor solubility, limited selectivity, and systemic cytotoxicity. It is well documented that PNPs accumulate in the tumor site in a high concentration by utilizing EPR. The main challenge against passively delivered PNPs is that angiogenesis is not uniformly distributed throughout the tumor leading to a disproportional distribution of NPs by EPR [41]. The active targeting based on the microenvironmental difference between cancer and healthy cells is crucial to add selectivity to EPR and overcome its limitations. One such difference is the expression levels of CD44 [42]. CD44 is a cell surface glycoprotein that is overexpressed and intensively involved in TNBC carcinogenesis [22,43]. Basal epithelial, basal mesenchymal TNBC, and CSCs are enriched with the CD44 receptor [44]. Therapeutic failure and cancer relapse are mainly due to the inability to eradicate CSCs [13,45]. Targeting of cancer cells and CSCs will effectively reduce tumor burden and relapse. CD44 has a tremendous binding affinity to HA, which attracts our attention to develop HA-based NPs leading to an increase in the affinity of NPs to cancer cells and CSCs and selective toxicity due to HA-CD44 receptor-mediated endocytosis [42,43]. In our study, HA-PNPs have proved their significance in developing CD44-T-PNPs with high cellular uptake in vitro, preferential tumor accumulation in vivo, and theranostic potential by enveloping both CFM 4.16 and S0456. That came in agreement with HA-SMA-NMS that effectively delivered CDF to the aggressive CD44+ stem-like pancreatic cancer cells [46] and HA-TPGS-DOX that increased doxorubicin cytotoxicity in MCF-7/ADR [47]. Adding vitamin E-TPGS improved Genexol-PM uptake and PTX cytotoxicity due to the enhancement of membrane fluidity and MDR inhibition; the IC 50 was reduced by 4.4 folds compared to Genexol-PM [40].
Many physiochemical characters of NPs affect their cellular interaction, such as shape, size, surface charge, and hydrophobicity. The study has opted for spherical with a smooth surface, <100 nm, slightly anionic, and water-soluble, which have proven ideal for use in the drug delivery system (DDS). Most of the developed NPs applied for DDS are spherical due to their being easy manufacting.. Even many studies showed that rod or disc NPs have a more favorable effect than spherical, there are contradictory studies [48]. This contradiction is owing to differences in the material composite, tested cell lines, and analyzing techniques [49][50][51]. Another reason why spherical-shaped NPs have been preferred is their lower reactivity and toxicity than fiber-shaped NPs [52]. In addition, whereas the non-spherical ones tumble with the flow, spherical shaped NPs are known for their ease of motion [53]. Furthermore, PNPs' smooth surface have the ability to reduce the phagocytosis process [54] and deposition rate [55]. The size of NPs is a determent factor for its clinical application. Our PNPs are able to escape glomerular filtration (<5 nm) and trapping by RES (>150 nm) [56]. Also, it is in the favorable size range for cellular uptake and extravasation via EPR. As the cut-off size of the endothelial gaps in tumor blood vessels ranges from 200 nm to 1.2 µm based on tumor type; therefore, NPs ≤ 200 nm are widely used for passive and active targeting of the tumor [57]. Controlling NPs size is critical for reducing genotoxicity (∼10 nm) and cytotoxicity to healthy cells [58]. NPs surface charge plays a vital role in drug loading, circulation time, cellular uptake, cellular cytotoxicity, NPs stability, and clearance by RES. The cationic NPs have many safety concerns, as they are strongly attracted to the negatively charged cell membranes leading to destabilization of cell membranes, leakage of cytoplasm, and, subsequently, cell lysis. Damage includes the endothelial lining of blood vessels, RBCs, and healthy cells. Elimination of NPs by RES based on their zeta potential is a controversial issue. It is generally accepted that neutral or slightly anionic NPs are preferred for their safe parenteral administration, lower systemic toxicity, higher tumor accumulation, prolonged circulatory lifetime, and less off-target uptake [53,56,59,60]. Our hydrophilic PNPs are far more vulnerable to immune detection as the hydrophilic NPs repel opsonions that reduce their recognition by mononuclear phagocyte system (MPS) and increase their circulatory time [49,61].
NT-PNPs and CD44-T-PNPs have an adequate LC% with respect to the polymer amount compared to other polymeric NPs preparation [62]. As it is well established that TNBC has tremendous esterase and hyaluronidase activity [63,64]. Both PNPs are esterase-responsive while only CD44-T-PNPs are additionally hyalu-ronidase-responsive. The carbonic ester bonds and the glycosidic bonds in the NPs will be disassembled by intracellular esterase and hyaluronidase, followed by releasing the antineoplastic agent. This will increase tumor specificity, reduce off-target toxicity, prevent premature drug release and circulation stability [63]. Our PNPs physicochemical properties are consistent with the marketed Genexol-PM ® that had a smooth spherical shape, −4.36 mv,16.67% LC, and two folds reduction in IC 50 compared to free PTX in A549 [40].
The current study has illustrated a promising synergism supported by the in vitro and in vivo anticancer activity. The co-treatment of momelotinib (MMB) and CFM-4.16 increases the downregulation of P-STAT3 accompanied by the upregulation of CARP-1, especially in T-combo. STAT3, particularly phosphorylated by JAK2, is one of breast cancer clinical significance [11]. STAT3 enhances cell proliferation by activating cyclin-dependent kinases (CDKs) by upregulation of cyclin D2 and downregulation of p21. STAT3 induces transcription of hypoxia-inducible factor (HIF-1a). STAT3/HIF-1a axis plays a crucial role in adapting tumor cells to the hypoxic environment associated with cancer progression. Also, STAT3 leads to overexpression of VEGF responsible for angiogenesis. Tumor invasion and metastasis are under the regulation of STAT3 by different mechanisms such as; induction of transcription of matrix-degrading enzymes such as matrix metalloproteinase and activation of epithelial to mesenchymal transition (EMT). STAT3 inhibition, either by knocking down or by pharmacological inhibitors, was found to suppress tumor invasion and metastasis in vivo and in vitro [65]. Inhibition of the JAK/STAT pathway has increased the sensitivity of resistant breast cancer cells to doxorubicin [66]. Worth emphasizing is, JAK2 /STAT3 is a prerequisite for the maintenance and proliferation of CSC of breast cancer and developing of chemo-and radio-resistance [13,67]. So, the JAK2/STAT3 in CSC is a potential target for developing a successful strategy to improve breast cancer patients' therapeutic outcomes.
The study has revealed that the combination of MMB and CFM-4.16 has induced apoptosis via JAK2/STAT3 inhibition-mediated ROS generation. Recently it has been reported that P-STAT3 is inversely related to ROS production [68][69][70]. ROS is regarded as a double-edged sword in cancer cells as a slight increase of ROS leads to cancer initiation and progression, while high levels of ROS induce cell senescence. It is known that the level of cancer intrinsic ROS is relatively higher than that of normal cells; thus, increasing the ROS production by chemotherapy will effectively eradicate cancer cells, but it will be inadequate to trigger apoptosis in healthy cells with a low level of intrinsic ROS [71]. Redox imbalance induces apoptosis by disturbing mitochondrial membrane potential, enhanced mitochondrial membrane permeability to pro-apoptotic proteins, including cytochrome c. In the cytosol, cytochrome c binds to Apaf-1 to form an apoptosome, which in turn activates caspase-9. Overactivation of caspase-9, in addition to caspase-8, p38 MAPK, upregulation of CARP-1, and PARP cleavage, took place by CFM-4.16 [16,17]. Taken together, activate the caspase 3/7 cascade pathway that was confirmed in our results leading to DNA damage, cell shrinkage, and cellular detachment [72], which were proved by TUNEL, cellular viability, and morphology (Summarized in Figure S4).
Optical imaging by targeted NIR dye has been evolved to enable observation of cancer burden and progression under various therapeutic strategies and stages and rapid monitoring of molecular events occurring within cells. Rapid assessment of the therapeutic efficacy in vivo is highly needed. As in relatively slow-growing models, the caliper measurements are unable to detect the difference for several days. Also, in orthotopic, metastatic, and systemic models, the longitudinal measurements of tumor burden are not possible. In our attempt, we provided targeted theranostic NPs, which was able to deliver both CFM-4.16 and S0456 NIR dye to the tumor site with limited off-target distribution compared to the non-targeted one. The results also showed that CD44-T-PNPs had an excellent prolonged effect in vivo confirmed by delayed renal clearance to 72 h. Probably because HA surface modification could act as a protective coating that reduces NPs opsonization and immunogenicity, leading to escape catching by RES in the blood [73,74]. The water solubility of the NPs will overcome the low solubility of anticancer agents and provide safe bio-elimination of the NPs. To the best of our knowledge, there is no FDA approved theranostic for TNBC, which is still an essential need in the clinical setup [75]. The intrinsic properties of our PNPs pave its application as a treatment option for TNBC.

Materials (Cell Lines and Chemicals)
TNBC cell lines MDA-MB-231 and MDA-MB-468 have been used as in vitro and in vivo model for human TNBC overexpressing CD44 receptors. Both cell lines were cultured in high glucose DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cell culture was maintained at 37 • C and 5% CO 2 conditions. Momelotinib was purchased from Adooq Bioscience (Irvine, CA, USA). CFM-4.16 were synthesized as described before [17,76]. SMA (M = 1.6 kDa) and sodium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vitamin E TPGS was purchased from Antares Health Products, Inc. (Jonesborough, TN, USA). Hyaluronic acid (MW = 13 kDa) was purchased from CosChemSupply (Los Angeles, CA, USA). EDC was purchased from CovaChem (Loves Park, Illinois, USA). All the other reagents used were of analytical grade. Cell culture DMEM, FBS, penicillin-streptomycin were purchased from GIBCO (Waltham USA, MA, USA).

Screening of In Vitro Cell Viability (MTT Assay) and Combination Study
The cytotoxicity of CFM 4. 16  of each drug. The cells have been treated by the noted concentrations for 72 h, followed by viability measuring by MTT assay, as previously described. The combination index (CI) and dose reduction index (DRI) were calculated by COMPUSYN software (COMPUSYN Inc, Paramus, NJ, USA). The CI is a representative quantitative measurement for the degree of drug interaction; CI < 1, CI = 1, and CI > 1 indicate synergism, additivity, and antagonism, respectively. The DRI is an indicator of how many folds the dose of each drug in a synergistic combination is reduced compared to the required dose of each drug alone to obtain the same effect. SMA-TPGS and HA-SMA-TPGS were prepared according to our previously reported method [38]. For the synthesis, 30 mg HA and 70 mg TPGS were dissolved in 50 mL deionized water (DI), then 200 mg/5 mL NaHCO3 was added to the solution. Then the pH was adjusted to 8.9, and 105 mg SMA in 10 mL DMSO was added. The reaction was left overnight until the solution becomes clear. The only difference in the NT carrier is that HA was not initially added. Both SMA-TPGS (NT carrier) and HA-SMA-TPGS (T carrier) were purified by dialysis bag (MWCO 2 kDa) for 24 h, lyophilized then characterized by FTIR and 1 H-NMR.

Preparation and Characterization of CFM-4.16 Loaded Polymeric NPs (PNPs)
Loading of CFM-4.16 was carried out according to our reported method [76]. First, 50 mg of carrier polymer was dissolved in 50 mL of DI water. Then 15 mg/mL of CFM-4.16 dissolved in DMSO was added to the polymer solution. Then 20 mg of EDC was added, and the pH was adjusted to 5.0, then 11 each for 30 min. Finally, the pH was adjusted to 8.0, and the free CFM

Loading Capacity (LC%) and Encapsulation Efficiency (EE%)
The LC% and EE% of NT-PNPs and CD44-T-PNPs were measured by high-performance liquid chromatography (HPLC). The mobile phase consisted of acetonitrile 65%, methanol 20%, and 10 mM potassium dihydrogen phosphate (KH 2 PO 4 ) with (pH 2) 15%, and the readout wavelength was 309 nm. Briefly, 1 mg of each formulation/1ml DI water was prepared, then 100 µg, 50 µg, and 25 µg concentrations were prepared using the mobile phase as a diluent. The average of triplicate injections of each sample was used on the standard curve equation; then, the LC%, EE%, and yield were calculated as follows: Loading Capacity (LC%) = (Amount of CFM4. 16 (Table 2). The effect of the combination on the morphology and metastasis was studied on MDA-MB-231. The cells were plated at 90% confluence in 6 well plates and treated with the indicated concentrations of the noted compounds for the selected time points. The cells were then fixed with 70% ice-cold ethanol for 10 min and stained with 0.4% crystal violet for 1 h. The stain was poured off, and the plates were washed and dried at room temperature. The wells were photographed at 10x. The only difference in wound healing assay that each well was scratched with a sterile 200 µL micropipette tip after 24 h incubation. The wound margin was photographed by an EVOS FL Auto (Life Technologies) microscope at 10× magnification at 0, 24 , and 72 h of treatment.

Detection of ROS Generation
ROS generation was detected using H 2 DCFDA according to the manufacturer's instructions. Briefly, the cells were plated in 70% confluency in 6 well plates (MDA-MB-231 0.5 × 10 6 cells/well and MDA-MB-468 1 × 10 6 cells/well). After 24 h, the cells were treated with the indicated concentrations of the noted compounds for 12 h, and H 2 O 2 was used as control positive. The media was then removed, wells were washed with PBS, and stained with 5 µM H 2 DCFDA for 30 min. Finally, the wells were washed with PBS and imaged by (EVOS FL Auto, Life Technologies) microscope (10×) with 10 s elapsed time. The fluorescence intensity (excitation 485 nm; emission 530 nm) was measured by ZEN 2012 blue edition software, and the difference in ROS production was calculated by GraphPad Prism [77].

Caspase 3/7Activity Assay
Caspase 3/7 activity was measured using the Caspase-Glo ® 3/7 assay (Promega, Madison, WI, USA) according to the manufacturer's recommendations. Cells were seeded in 96 well plates; the next day, cells were treated with the indicated concentrations of the noted compounds for 24 h. Then Caspase-Glo 3/7 reagent was added, and the plates were incubated for 60 min. Luminescence was measured using the microplate reader, and significance calculated by GraphPad Prism.

Western Blot Analysis
The ability of our combination to restore the balance between the CARP-1 tumor suppressor gene and STAT3 oncogene was determined by western blot. The cells were plated in 70% confluency (1.5 × 10 6 /100 mm plate for MDA-MB-23 and 3 × 10 6 /100 mm plate for MDA-MB-468). After 24 h, the cells were treated with the indicated concentrations of the noted compounds for 12 and 48 h. The cells were harvested and lysed by RIPA buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) for 15 min at 4 • C. The lysates were then centrifuged at 12,000 rpm at 4 • C for 15 min. The supernatant was collected, and the protein concentration was determined by the Protein Assay Kit (Thermo Scientific). After protein normalization, 20 µg of protein extract from each sample was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 8%, followed by wet transferring to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) by standard procedures. The non-specific binding sites were blocked by 5% skimmed milk in 1× TBST for 1 h. Membranes were incubated with the noted dilution's of the primary antibodies, as shown in (Table S1) overnight at 4 • C followed by incubation with 1:10,000 horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 2 h at RT. The antigen-antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Biosciences, Little Chalfont, Buckinghamshire United Kingdom) and exposure to X-ray film (X-Omat, Kodak, Rochester, NY, USA). The same membranes were then re-probed with anti-GAPDH antibody as an internal control.

Animal Husbandry and Tumor Induction
Female mice were purchased from Jackson Laboratories, housed in a sterile environment on a standard 12 h light/dark cycle, and kept on regular rodent diet and water. All animal procedures were approved by the Wayne State Animal Care and IACUC committee in accordance with NIH guidelines. Mice were subcutaneously injected in their right flanks with MDA-MB-231(5.0 × 10 6 cells per mouse) suspended in Matrigel. Tumor growth was measured in two perpendicular directions weekly with a caliper. Tumor volumes were calculated using formula 0.5 × a × b 2 , where a is the measurement of the longest axis, and b is the other perpendicular axis. Tumors were allowed to grow for one month till the tumor became palpable with an average size of 431.5 mm 3 .

CD44 Expression in TNBC Bearing Mice Model by Immunohistochemistry
CD44 expression was studied after obtaining tumor from TNBC bearing mice. The tumor was sectioned to 5 µm paraffin-embedded tissue sections. These sections were permeabilized with (500 µL Triton X + 25 g BSA + 500 mL PBS) 5 min, three times. Then tumor sections were blocked by 5% BSA for 1 h. The tumor area was circled by immunopen and incubated with Alexa Fluor 488 anti-mouse/human CD44 antibody (Biolegend San Diego, CA, USA) overnight in the fridge. The next day, sections were washed, and nuclei were stained with Hoechst 33342 (1 µg/mL) for 15 min. Then sections were rewashed and dried, followed by adding the mounting media and coverslips. Imaging by confocal microscope at 10× and 63× oil immersion lens adjusted for the blue channel (352-461) for Hoechst stained nuclei and green channel for Alexa fluor anti-CD44 antibody (488-519).

TUNEL Assay
One of the critical hallmarks of late apoptosis is extensive genomic DNA fragmentation. This process generates multiple DNA double-strand breaks (DSBs) with accessible 3 -hydroxyl (3 -OH) groups that allow apoptosis detection by TUNEL assay. For this study, animals were divided into four groups; control negative, momelotinib 5 mg/kg, CFM-4. 16 15 mg/kg, and T combo group received 5 mg/kg momelotinib + 15 mg/kg CD44-T-PNPs. Animals received two doses every other day, then all animals were sacrificed, and the tumor tissues were sent to the Biobank core facility for paraffin-embedded tissue sectioning for TUNEL assay. The treatment with elevated apoptosis is indicated by increased brown staining or dark-brown spots. This short-term study was carried to see the capability of the CD44-T-PNPs combo to enhance the onset of apoptosis when compared with individual drugs.

NIR Imaging and Biodistribution Study
The feasibility of using the NT-PNPs and CD44-T-PNPs as a theranostic tool was tested in TNBC-bearing animal model. Animals were divided into three groups; free S0456 NIR dye, NT-PNPs/S0456 conjugate, and CD44-T-PNPs/S0456 conjugate. Conjugation of both formulations with S0456 NIR dye was carried as following; 10 mg of the already prepared NT-PNPs and CD44-T-PNPs were dissolved in 1:1 mixture of chloroform and methanol. Then 1 mg of S0456 NIR dye dissolved in 1 mL DI water was added to the chloroformmethanol mixture. Then chloroform and methanol were evaporated by Rotavapor (R-205, Buchi, Flawil, Switzerland). The solutions were dialyzed using a dialysis bag (MWCO 3.5 kDa) for 3 h then lyophilized. The incorporation of S0456 NIR dye to PNPs was measured by UV-spectrophotometer (Hitachi 2910) adjusted to 789 nm wavelength and calculated based on the standard curve equation. Mice were injected in the tail vein with 10 nmol/mouse of free S0456, NT-PNPs/S0456, and CD44-T-PNPs/S0456. Mice were imaged at 24 and 72 h post-injection using an In Vivo MS FX Extreme system (Carestream, San Diego, CA, USA); light source: 400 W xenon, monochrome interlined, fixed lens (10×), cooled (−60 • C), CCD camera, with 750 nm-830 nm wavelength for fluorescence, and X-ray images were captured. Both fluorescence and X-ray images of the mouse were merged to demonstrate the localization of NPs. Importantly, to understand the behavior and distribution of PNPs to tumor vs. healthy tissues, the NIR fluorescence of organ bio-distribution was carried out 72 h post-injection.

Conclusions
Despite the undeniable achievements in oncotherapy, cancers' unsatisfactory survival rates remain an issue of concern and a challenge that scientific research is yet, to overcome. Even though chemotherapy has proven successful as a therapeutic paradigm, numerous elements deny the possibility of depending solely on it. For instance, poor solubility and off-targeting biodistribution are among the many existing challenges that limit cancer chemotherapy's efficacy. Not only does this pr5event parenteral administration and leads to chemotherapeutics toxicity, the rapid evoking of chemotherapeutic resistance also leads to tumor relapse. Thereupon, in an attempt to address these challenges, NPs drug delivery systems (DDS) and combination strategies are currently being investigated. In this study combination of momelotinib with the CD44 directed CFM-4.16 PNPs was able to wipe out cancer cells efficiently compared to individual drugs. The developed nanomaterials' intrinsic properties such as biodegradability, water-solubility, loading contents, and cellular internalization potentiate its application as an excellent DDS for hydrophobic CFM-4.16 and hydrophilic S0456 NIR dye. The study has opted for PNPs of smooth spherical shape, acceptable size (<100 nm), a slightly negative charge, and selective tumor uptake. Upon administration, the CD44-T-PNPs ester backbone was hydrolyzed into non-toxic products with gradual releasing of the CFM-4.16 molecules and S0456 in the tumor site with low off-target distribution, which pave its application as a promising tool for the theranostic purpose.

Data Availability Statement:
The data presented in this study are available in this article (and supplementary material).