**4. Targeting GAGs in Cancer—New Prospective**

*4.1. Targeting Heparan Sulfate/Heparin*

degrees of acetylation 0.8, 2.1, 2.6 acetyl groups per unit (2 glucose rings)

HA super-paramagnetic iron

HS, expressed by all mammalian cells in homeostasis [31], has been determined to be the most complex GAG [19]. This highly variable GAG is critical in cellular signaling and extensively remodeled during cancer progression. In its natural state, Hep is a heterogeneous mixture composed of polysaccharide chains that exhibit varying lengths and different sulfation patterns. Hep, compared to HS, is more homogeneous and its main function is storage. HS and Hep chains can establish specific interactions with various protein mediators regulating critical cellular signaling [18]. The affinity of HS/Hep chains to proteins such as growth factors seems to be significantly affected by their sulfation status and resulting electrostatic interactions [157–159]. Moreover, analysis by the polyelectrolyte theory demonstrated that the binding of FGF-2 to Hep is primarily accomplished through the more specific nonionic interactions, such as van der Waals packing and hydrogen bonding [201]. Therefore, inherent properties of the GAG chains need to be taken into account when designing novel drug carriers [157–159].

oxide Doxorubicin breast [194]

HA-titanium dioxide Cisplatin ovarian [195]

Doxorubicin cervical [188]

To date, more than 400 HS-binding proteins have been identified, including cytokines, growth factors, chemokines, ECM proteins, as well as enzymes and enzyme inhibitors [18]. Thus, the targeting of HS protein interactions is an essential developing therapy approach.

The strategies that have been examined for cancer-oriented therapy are based on (i) the utilization of GAG mimetics as competitive agents to block HS–protein interactions (ii) the utilization of enzymatic methods to cleave or modify HS to inhibit HS–protein interactions.

The utilization of unfractionated Hep and LMWHs is standard clinical practice for the protection against venous thromboembolism in cancer patients [202]. This clinical practice's implementation has also demonstrated a beneficial effect of Hep on cancer patient survival discrete from its anticoagulant properties [203]. Indeed, Hep has now been recognized as a multifunctional drug [50]. Hep mimetics are commonly described as synthetic or semisynthetic compounds that are anionic, usually highly sulfated, and structurally defined as distinct GAG analogs [204].

Research efforts focused on the synthesis of Hep derivatives with attenuated polypharmacy traits and anticoagulant activity, exhibiting enhanced potency and specificity while downregulating unwanted side effects, e.g., anticoagulation [204]. This approach has been facilitated by significant development in carbohydrate synthesis, including one-pot multi-step procedures and coupling reactions, enabling the synthesis of complex oligosaccharides [205].

A recently synthesized, multitargeting Hep-based mimetic, necuparanib, was shown to attenuate pancreatic cancer tumor cell growth and invasion in a three-dimensional (3D) culture model. In contrast, in vivo, it facilitated survival and attenuated the metastatic ability of pancreatic cancer cells. Furthermore, the proteomic analysis demonstrated that necuparinib, among others, targeted ECM-originating mediators, well established to affect cancer cell growth and metastasis. Specifically, necuparanib attenuated the expression of metalloproteinase 1 (MMP1) and facilitated the expression of tissue inhibitor of metalloproteinase 3 (TIMP3) in the 3D pancreatic cancer model [206]. Moreover, the levels of TIMP3 in the plasma of patients with metastatic pancreatic cancer who were participating in a phase I/II study treatment with necuparanib plus standard therapy were found to be substantially enhanced [206].

A crucial therapeutic target is cancer-associated angiogenesis. Both fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) can form ternary complexes with HS and their respective cell-membrane receptors, initiating signaling cascades that facilitate angiogenesis [207]. These growth factors are characterized as important cancer therapy targets with Hep mimetics' possible implementation [208,209]. The D-mannosebased sulfated oligosaccharide mixture, PI-88 (Muparfostat) is one such inhibitor. It is developed from the oligosaccharide phosphate fraction obtained from the extracellular phosphomannan, initially derived from the yeast *Pichia (Hansenula) holstii* (NRRL Y-2448) and subsequently extensively sulfated [210,211].

Modified LMWH functionalized by polystyrene (NAC-HCPS) exhibited increased affinity to HS binding growth factors and attenuated anticoagulant properties, decreased endothelial cell growth, and formation of endothelial tubes [212]. Moreover, SST0001 Hep derivatives, characterized by 100% N-acetylated, 25% glycol split Hep SST0001 (100NA-ROH, roneparstat), efficiently reduced FGF2-mediated proliferation of endothelial and lymphoid cells and displayed a limited capacity to release FGF from the ECM. This effect is associated with the N-acetylation of GlcN.SST0001 and was also reported to counteract human sarcoma cell invasion induced by exogenous FGF2 [213]. Interestingly, Hep is actively uptaken by melanoma cells and affects their migration and adhesion [214].

The disadvantages of using Hep derivatives, discussed above, are mostly correlated to the intrinsic Hep anticoagulant properties to initiate severe hemorrhagic effects.
