Differentiating Co-Delivery of Bisphosphonate and Simvastatin by Self-Healing Hyaluronan Hydrogel Formed by Orthogonal "Clicks": An In-Vitro Assessment

Abstract

Due to its unique properties resembling living tissues, hydrogels are attractive carriers for the localized and targeted delivery of various drugs. Drug release kinetics from hydrogels are commonly controlled by network properties and the drug-network interactions. However, and simultaneously, the programmable delivery of multiple drugs with opposing properties (hydrophilicity, molecular weight, etc.) from hydrogels with determined network properties is still challenging. Herein, we describe the preparation of injectable self-healing hyaluronic acid (HA) hydrogels that release hydrophobic simvastatin and hydrophilic aminobisphosphonate (BP) drugs independently in response to acidic and thiol-containing microenvironments, respectively. We apply a prodrug strategy to BP by conjugating it to HA via a self-immolative disulfide linker that is stable in the blood plasma and is cleavable in the cytoplasm. Moreover, we utilize HA-linked BP ligands to reversibly bind Ca²⁺ ions and form coordination hydrogels. Hydrazone coupling of hydrophobic ligands to HA permits the encapsulation of simvastatin molecules in the resulting amphiphilic HA derivative and the subsequent acid-triggered release of the drug. The conjugation of BP and hydrophobic ligands to HA enables preparation of both bulk self-healing hydrogels and nanogels. Moreover, the developed hydrogel system is shown to be multi-responsive by applying orthogonally cleavable linkers. The presented hydrogel is a potential candidate for the combination treatment of osteoporosis and bone cancers as well as for bone tissue regeneration since it can deliver bone anabolic and anti-catabolic agents in response to bone diseases microenvironments.

Keywords: bisphosphonate; hyaluronan; hydrogel; orthogonal reactions; prodrug; simvastatin.

Figure 1

The nanomedicine platform based on the self-assembly of hyaluronic acid conjugated to bisphosphonates and hydrophobic ligands through orthogonally releasable linkers. Hydrophobic encapsulation of bone anabolic agents occurs during the self-assembly and provide nanoparticles with a dual mode of bioactivity.

Scheme 1

Synthesis of heterobifunctional linker 3 and hyaluronan-bisphosphonate prodrug HA-//-BP with a thiol-triggered release mechanism.

Figure 2

(a) Image of hydrogel formed by coordination cross-linking of HA-//-BP derivative by Ca²⁺ ions. (b) Frequency oscillation sweep of HA-//-BP•Ca²⁺ hydrogel. (c) Strain oscillation sweep of HA-//-BP•Ca²⁺ hydrogel. (d) Time oscillation sweep of HA-//-BP•Ca²⁺ hydrogel upon alteration of strain between low (1%) and high (300%) values. Concentrations of HA-//-BP and Ca²⁺ ions in HA-//-BP•Ca²⁺ hydrogel were 3% and 0.2 M, respectively.

Scheme 2

(a) Synthesis of the model prodrug 5 carrying 2-dithiopyridyl groups and (b) its further conjugation to hyaluronan to obtain HA derivative with permanently attached BP groups and releasable prodrug moieties (BP-HA-//-DN).

Figure 3

Two types of hyaluronan-bisphosphonate derivatives (HA-//-BP and BP-HA-//-D where D stands for a drug and -//- stands for a releasable self-immolative linker) and the respective injectable hydrogel-based drug delivery systems: (a) Intact bisphosphonate-releasing hydrogel that is decomposed upon action of thiols and (b) hydrogel-linked prodrug that releases amine-bearing drugs upon action of thiols.

Figure 4

(a) Absolute amount and (b) percentage of N-(6-aminohexyl)-2,4-dinitroaniline (DN) released from Ca²⁺•BP-HA-//-DN hydrogel upon incubation in 0.17 M NaCl containing 2.5 mM CaCl₂ (pH 7.4). The red curve corresponds to the release when 20 mM DTT was present in the incubation medium. The green curve corresponds to the release when no DTT was present in the release medium.

Scheme 3

(a) Synthesis of aldehyde-modified DN derivative and hyaluronic acid bearing hydrazide and releasable BP groups (hy-HA-//-BP). (b) Use of hy-HA-//-BP derivative for the preparation of hydrazone and coordination hydrogels as well as for generation of amphiphilic HA derivative bearing hydrophobic DN and hydrophilic BP groups (DN-hyd-HA-//-BP).

Figure 5

¹H-NMR spectrum of DN-hyd-HA-//-BP derivative.

Figure 6

(a) Amount of DN derivative 6 released upon incubation of SIM@DN-hyd-HA-//-BP in either 0.1 M NaOAc/AcOH pH = 5.0 buffer (red curve) or 1 × PBS buffer pH = 7.4 (blue curve). (b) Amount of simvastatin (SIM) released upon incubation of SIM@DN-hyd-HA-//-BP in either 0.1 M NaOAc/AcOH pH = 7.4 buffer (green curve) or 1 × PBS buffer pH = 7.4 (brown curve).