ArticlePDF AvailableLiterature Review

Hyaluronic Acid‐Based Bioconjugate Systems, Scaffolds, and Their Therapeutic Potential

Advanced Healthcare Materials

April 2023
12(20):e2203104

DOI:10.1002/adhm.202203104

Authors:

Niranjan Kotla

Novartis Institutes for BioMedical Research (NIBR)_Switzerland

Isma Liza Mohd Isa

Universiti Kebangsaan Malaysia

Aitor Larrañaga

Universidad del País Vasco / Euskal Herriko Unibertsitatea

Balaji Maddiboyina

Freyr Global Regulatory Solutions & Services

Show all 7 authorsHide

In recent years, the development of hyaluronic acid or hyaluronan (HA) based scaffolds, medical devices, bioconjugate systems have expanded into a broad range of research and clinical applications. Research findings over the last two decades suggest that the abundance of HA in most mammalian tissues with distinctive biological roles and chemical simplicity for modifications have made it an attractive material with a rapidly growing global market. Besides its use as native forms, HA has received much interest on so‐called “HA‐bioconjugates” and “modified‐HA systems”. In this review, the importance of chemical modifications of HA, underlying rationale approaches, and various advancements of bioconjugate derivatives with their potential physicochemical, and pharmacological advantages are summarized. This review also highlights the current and emerging HA‐based conjugates of small molecules, macromolecules, crosslinked systems, and surface coating strategies with their biological implications, including their potentials and key challenges discussed in detail.

a,b). Graphical data showing the number of articles published from 1970 to present on “hyaluronan” and “hyaluronic acid conjugates”. Source: PubMed, last accessed February 2023. c) Number reported of clinical trials on hyaluronan (where, Early Phase I: exploratory trials before Phase I with very limited human exposure; Phase IV: postmarketing surveillance; not applicable: trials without FDA‐defined phases, such as devices or behavioral intervention trials. Source: https://clinicaltrials.gov/, accessed on February 2023).

…

a) HA disaccharide unit chemical structure and its structural, physical, and biological properties. b) The regulation of HA synthesis by the HA synthase (HAS 1, 2, and 3 enzymes) and degradation mainly by hyaluronidase enzyme and nonenzymatic (ROS – reactive oxygen species, hydrolysis, and thermal degradation) approaches. c) HA biopolymeric networks forming noncovalent assemblies with HA binding proteoglycans in the tissue microenvironment(s), the possible associated surface receptor interactions, and downstream signaling mechanisms.

…

Schematic illustration of various chemical modifications of HA including esterification, amidation, and crosslinking on carboxylic group of each monomer; several OH functionalization on hydroxyl groups (here hyaluronic acid shown as HA or AH).

…

Chemical structures of various reported small molecule drug–HA conjugates. a–j) HA biopolymer conjugation to model anti‐inflammatory, anticancer, local‐anesthetic, and steroid small molecule drugs. HA‐small molecule drug conjugates have been employed to improve targetability, solubility, cellular absorption, longer half‐life, increase the delivery system stability, and extended release with combinatorial biological functions.

…

+10

a,b) Schematic synthesis illustration of polypseudorotaxane and polydopamine‐based hydrogel structures, as well as the design and fabrication of a PDM‐loaded HD‐PEG/‐CD hydrogel construct for subcutaneous, long‐acting injectable delivery of donepezil. Reproduced with permission.[⁴⁶] Copyright 2021, Elsevier.

…

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REVIEW

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Hyaluronic Acid-Based Bioconjugate Systems, Scaﬀolds,

and Their Therapeutic Potential

Niranjan G. Kotla,* Isma Liza Mohd Isa, Aitor Larrañaga, Balaji Maddiboyina,

Samantha K. Swamy, Gandhi Sivaraman,* and Praveen K. Vemula*

In recent years, the development of hyaluronic acid or hyaluronan (HA) based

scaﬀolds, medical devices, bioconjugate systems have expanded into a broad

range of research and clinical applications. Research ﬁndings over the last two

decades suggest that the abundance of HA in most mammalian tissues with

distinctive biological roles and chemical simplicity for modiﬁcations have

made it an attractive material with a rapidly growing global market. Besides

its use as native forms, HA has received much interest on so-called

“HA-bioconjugates” and “modiﬁed-HA systems”. In this review, the

importance of chemical modiﬁcations of HA, underlying rationale approaches,

and various advancements of bioconjugate derivatives with their potential

physicochemical, and pharmacological advantages are summarized. This

review also highlights the current and emerging HA-based conjugates of

small molecules, macromolecules, crosslinked systems, and surface coating

strategies with their biological implications, including their potentials and key

challenges discussed in detail.

1. Introduction

Hyaluronic acid (HA) is a naturally occurring nonprotein, lin-

ear, nonsulfated glycosaminoglycan, a major component of the

extracellular matrix (ECM) of several biological tissues. Over

the last few decades, native HA and modiﬁed-HA systems have

witnessed an increased progression due to their promising

N. G. Kotla, P. K. Vemula

Institute for Stem Cell Science and Regenerative Medicine (inStem)

Bangalore, Karnataka 560065, India

E-mail: niranjan.kotla5@gmail.com; praveenv@instem.res.in

I. L. Mohd Isa

Department of Anatomy

Faculty of Medicine

Universiti Kebangsaan Malaysia

Kuala Lumpur 56000, Malaysia

A. Larrañaga

Department of Mining-Metallurgy Engineering and Materials Science

POLYMAT

Faculty of Engineering

University of the Basque Country (UPV/EHU)

Bilbao 48013, Spain

The ORCID identiﬁcation number(s) for the author(s) of this article

can be found under https://doi.org/10.1002/adhm.202203104

DOI: 10.1002/adhm.202203104

multifaceted biophysical, biochemi-

cal, viscoelastic, and tissue remodeling

properties.[– ] A fairly accurate literature

search (in between  and Present)

on the PubMed website using the key-

words “hyaluronan,” “hyaluronic acid

conjugates,” gave total   and 

hits, respectively (as shown in (Figure

1a,b). Similarly, there are more than 

clinical trial reports available (Figure c)

(https://clinicaltrials.gov/, accessed on

February, ). Noticeably, this search

can be considered as a rough and not a

bibliography of a speciﬁc research topic.

Nevertheless, the number of published

research reports suggests that after an

initial pioneering period, HA has intrigued

biomedical researchers ever since it was

discovered because of the breadth of its

unique biophysical, mechanical and bio-

logical functionalities. According to the

current projections, the worldwide HA

market will be worth approximately USD . billion by ,

as HA-based products are rapidly evolving, especially in der-

mal, cosmetic, ocular, mucosal, bone, and other biomedical

applications.[]

Recently, the U.S. Food and Drug Administration (FDA) has

proposed to reclassify HA-based compounds, devices, products,

B. Maddiboyina

Department of Medical Writing

Freyr Solu ti on s

Hyderabad, Telangana 500081, India

S. K. Swamy

Thrombosis Research Center (TREC)

Department of Clinical Medicine

UiT-The Arctic University of Norway

Tromsø 9037, Norway

G. Sivaraman

Department of Chemistry

Gandhigram Rural Institute (Deemed to be University)

Gandhigram, Tamil Nadu 624302, India

E-mail: raman474@gmail.com

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1398 2451

5297

10390

21022

9342

Number of publications

Hyaluro

anan

310 51 116

919

465

Number of publications

Hyaluronic acid conjugates

Early Phase I (12)

Phase I (37)

Phase II (65)

Phase III (66)

Phase IV (90)

Not applicable (338)

Total=608

Figure 1. a,b). Graphical data showing the number of articles published from 1970 to present on “hyaluronan” and “hyaluronic acid conjugates”. Source:

PubMed, last accessed February 2023. c) Number reported of clinical trials on hyaluronan (where, Early Phase I: exploratory trials before Phase I with

very limited human exposure; Phase IV: postmarketing surveillance; not applicable: trials without FDA-deﬁned phases, such as devices or behavioral

intervention trials. Source: https://clinicaltrials.gov/, accessed on February 2023).

and delivery systems as therapeutic drugs by citing scientiﬁc tes-

timony, especially as HA provides pain relief eects (for, e.g., in

joint inﬂammation) through its biochemical activities in the liv-

ing systems;[, ] however, recent clinical studies data did not show

eective therapeutic beneﬁt of HA injections in osteoarthritis

therapy as that of placebo.[] Henceforth, future HA-based prod-

ucts will have more regulatory scrutiny while entering into clini-

cal practice.[, ]

The recent literature reports have shown that HA molecular

mass, modiﬁed-HA systems inﬂuence various cellular, molec-

ular signaling mechanisms, matrix remodeling, and tissue

homeostasis.[] Besides, thanks to the unique potential thera-

peutic properties of HA with dierent biological role(s) in ECM

organization,[, ] cell proliferation,[, ] dierentiation,[– ]

wound healing processes,[, ] immunomodulation,[]

inﬂammation,[, ] modulating epithelial barrier,[]

viscosupplementation,[, ] and various cellular signaling pro-

cesses associated with tissue homeostasis, matrix remodeling,

and others.[,,, ]

The present review primarily describes the functional, bio-

chemical properties, and limitations/challenges of native HA (bi-

ologically present HA). After that, from taking the existing liter-

ature into account, we put forward the criterion for developing

modiﬁed-HA-based scaolds, HA-bioconjugate derivatives, and

their potential beneﬁts for various biomedical applications. Be-

sides, the purpose of the review is to give a clear picture of ad-

vanced ﬁndings of modiﬁed-HA scaolds, with recently reported

applications of HA-based conjugates including small and macro-

molecule conjugates, crosslinked systems, cell-based HA scaf-

folds, HA-functionalized inorganic systems, and HA biocoating

approaches are also discussed in detail.

2. Structural, Functional Aspects of HA and the

Rationale for Bioconjugation of Therapeutics

The native form of linear, long-chain HA biopolymer has repeat-

ing disaccharide units of 𝛽-,--glucuronic acid and 𝛽-,-N-

acetyl--glucosamine bound with 𝛽-glycosidic linkages (Figure

2a). HA exists in ionized or salt forms at physiological pH, such

as sodium hyaluronate. Due to the presence of carboxylic groups

on each monomer, the molecule has an anionic property, making

it very hydrophilic; besides, the abundance of negatively charged

hydroxyl groups of HA can bind and hold a high number of wa-

ter molecules.[] In biological systems, the aforementioned re-

peating disaccharides can be >  units to form high molec-

ular weight (more than  MDa) polymeric networks.[, ] Litera-

ture evidences suggest that HA biopolymer single disaccharides’

average length is ≈ nm (also reported as Kuhn length); as an,

e.g., HA polymer with   repetitions may be stretched out to

≈ μm.[] These HA chains in the cellular matrix build a dense,

brush like structure called pericellular matrix (PCM), in which

various proteoglycans attach, and modify the structure of the HA

chains. The composition and size of the PCM layer dier from

tissue to tissue based on their biological functionality.[]

In particular, these network structures further form noncova-

lent dense assemblies with HA binding proteoglycans (aggrecan,

chondroitin sulfate, versican, etc.) based on the type of tissue

microenvironment and ECM composition.[, ] HA synthesis

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Figure 2. a) HA disaccharide unit chemical structure and its structural, physical, and biological properties. b) The regulation of HA synthesis by the HA

synthase (HAS 1, 2, and 3 enzymes) and degradation mainly by hyaluronidase enzyme and nonenzymatic (ROS – reactive oxygen species, hydrolysis,

and thermal degradation) approaches. c) HA biopolymeric networks forming noncovalent assemblies with HA binding proteoglycans in the tissue

microenvironment(s), the possible associated surface receptor interactions, and downstream signaling mechanisms.

(regulated by the HA synthase (HAS) , , and  enzymes) and

degradation (enzymatic, nonenzymatic) solely depend on the tis-

sue microenvironment; the physiological concentrations of HA

in tissues are essential to perform its regulatory functions. Each

enzyme has speciﬁc functionality in synthesizing appropriate

size range of HA polymer chains. HAS mainly produces the

small chains (.–. MDa), and HAS, HAS produce longer

chains (. to  MDa or more), achieving polymer chains up to

 μminlength

[] (Figure b,c).

The hydrodynamic radius or size of the HA polymer depends

on the molecular weight; as the HA molecular weight increases

( to  kDa) the hydrodynamic radius increases from  to

 nm.[] HA’s biophysical features (such as molecular weight,

chain length, crosslinking nature, viscoelasticity, adhesivity, cell-

tissue speciﬁc mechanics, hydrodynamic and degradation prop-

erties) can have a signiﬁcant impact on cell to tissue mechan-

ics, ECM organization, tissue homeostasis, and alter healthy ver-

sus disease condition. As a result, along with other proteogly-

cans, HA is reported to be an element for a number of healthy

and disease-related processes.[] The biophysical and mechani-

cal characteristics of HA can be tunable and dependant on dier-

ent modiﬁed-HA forms including bioconjugate systems.

The growing evidence reveals that HA plays a key role in sev-

eral biophysical and biomechanical events in the ECM by binding

with matrix components and cell receptor interactions (speciﬁc

and nonspeciﬁc), especially with CD, RHAMM/CD,

LYVE-, TLRs, HARE, layilin, and others.[] CD involved in

HA-internalization and alter the ECM components degradation,

angiogenesis, cell adhesion, proliferation, and migration; notably

the immune cells activation, tracking, apoptosis and, impor-

tantly immunotolerance.[] Upon interaction of HA, these recep-

tors induce several intracellular signaling processes including

cell growth, tissue homeostasis, mechanotransduction, tumor

metastasis, inﬂammation, immunomodulation, and others (see

Figure ). Regardless of the successful application of unmodiﬁed-

HA, HA-based systems, its short half-life, poor mechanical

stability, uncontrolled mechanical, swift degradation behavior,

and poor adhesivity properties should be overcome for long-term

biomedical and clinical applications. Biopolymers, including HA

with biochemical stimuli mechanisms, provide essential clues

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Cross linking

Figure 3. Schematic illustration of various chemical modiﬁcations of HA including esteriﬁcation, amidation, and crosslinking on carboxylic group of

each monomer; several OH functionalization on hydroxyl groups (here hyaluronic acid shown as HA or AH).

toward developing more eective modiﬁed-HA-based conjugates

or scaolds, which require consideration of various synthesis

approaches to develop standard modiﬁed-HA-based systems

with enhanced pharmacological, and therapeutic beneﬁts.

3. Modiﬁcations of HA and Their Derivatives

HA biopolymer is susceptible to a variety of chemical modiﬁ-

cations through three orthogonal functional moieties (hydroxyl,

carboxyl, and amide), facilitating its use for numerous applica-

tions requiring bioconjugation, chemical crosslinking, and sur-

face modiﬁcations of the delivery system.[] Chemical modiﬁ-

cation of HA can be accomplished in two ways via crosslink-

ing or conjugation methods. The chemical modiﬁcations of HA

have been primarily carried out at dierent sites, including car-

boxyl groups of the 𝛽--glucuronic acid (GlcUA), hydroxyl groups

of the N-acetyl-𝛽--glucosamine (GlcNAc) residue, and the re-

ducing end of the disaccharide repeating unit. It is also possi-

ble to generate primary amino groups on the HA polymer chain

by deacetylation of GlcNAc for chemical conjugation of the acid

derivatives via amide formation. The chemical reactions of HA

are carried out either in aqueous or organic solvents such as

dimethylsulfoxide or dimethylformamide.[]

More importantly, the carboxylic acid group of HA is

frequently reacted with primary amine derivatives using

carbodiimide-mediated reactions under aqueous conditions.[]

Due to its water solubility, -ethyl--[-(dimethylamino)-propyl]-

carbodiimide (EDC) is the most often used carbodiimide.

Danishefsky and Siskovic were the ﬁrst to use EDC to activate

the carboxyl groups of polysaccharides (pH .), including

HA, and then interacted with an amino acid ester to form

amides.[] Several publications have described Ugi condensa-

tion for HA crosslinking, which involves employing diamine as

a crosslinker to produce diamide connections between polysac-

charide chains.[, ] The synthesis of methyl ester of HA using

trimethylsilyl diazomethane (TMSD) as the carboxylic group

activator was reported, in which TMSD reacts with HA to form

an intermediate followed by acetic acid reaction to recover

the methyl ester.[] Several publications have documented the

interaction between HA and glycidyl methacrylate to produce

methacrylated HA.[, ] Besides, the hydroxyl groups of HA have

been used for esteriﬁcation (using anhydrides or acid chlorides)

or ether formation (using epoxides, ethylene sulﬁdes, or divinyl

sulfones).[] Figure 3 illustrates the various possible chemical

modiﬁcations of HA.

A large number of HA derivatives have been documented in

the literature, each of which is intended for a speciﬁc use, in par-

ticular various delivery systems (mentioned in Figure 4 and in

the next sections). More importantly, HA is a natural ligand of

several cell surface receptors as mentioned in the previous sec-

tion including CD, which overexpressed in several types of can-

cer cells. For example, conjugation of anticancer drugs to HA

signiﬁcantly improves the solubility, stability, and targetability

to cancer tissues (brieﬂy discussed in further sections). Besides,

HA is a unique biopolymer (with unique properties of water-

solubility, biocompatibility, biodegradability, the ability to target

to cells, and presence of multiple functional groups) that com-

bines many favorable attributes as a carrier for the conjugation-

based therapeutics delivery and imaging agents for biomedical

uses. In the next sections, we have provided the recent advance-

ments of various HA-based small, macromolecule conjugates,

crosslinked, and coating systems and their potential beneﬁts for

various biomedical applications.

4. HA-Based Small Molecule Conjugates

Since the beginning of modern-day medicine, small-molecule

drugs have dominated the pharmaceutical industry with several

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Figure 4. Chemical structures of various reported small molecule drug–HA conjugates. a–j) HA biopolymer conjugation to model anti-inﬂammatory, an-

ticancer, local-anesthetic, and steroid small molecule drugs. HA-small molecule drug conjugates have been employed to improve targetability, solubility,

cellular absorption, longer half-life, increase the delivery system stability, and extended release with combinatorial biological functions.

blockbuster drug candidates in the recent years until today.[]

Some of the common challenges in developing broad range of

small molecule drug based therapeutics are lack of bioavailabil-

ity, targetability, low cellular uptake, rapid degradation/clearance,

etc. Thus, prodrugs, drug-polymer scaolds, and modiﬁed-drug

conjugates including HA-conjugate systems (along with its

added therapeutic beneﬁts) have been used to increase the tar-

getability of the drugs, with enhanced cellular uptake, prolonged

half-life, increased stability of the delivery systems, extended re-

lease with multiple functionalities. More recent studies attempt

to utilize the aforementioned properties of HA to develop HA-

based drug conjugates and to alter the biotherapeutic activity of

HA itself based on its range of available molecular weights.[, ]

In the below (Figure ), we have shown the reported small

molecule drug–HA conjugates investigated for various therapeu-

tic applications in particular anticancer therapies.

HA-conjugated paclitaxel has shown selective toxicity toward

various human cancer cell lines (breast, colon, and ovarian) that

are known to overexpress HA receptors. Conjugating hydropho-

bic anticancer drugs such as paclitaxel and -ethyl--hydroxy-

camptothecin (SN-) to HA signiﬁcantly helps to overcome the

limited aqueous solubility and stability of these chemotherapeu-

tic agents.[] By taking the advantage of receptors overexpressed

on cancer cells including CD and RHAMM, various hydropho-

bic anticancer agents (paclitaxel, docetaxel, etc.) conjugation to

HA have signiﬁcantly ameliorated the solubility, stability, and tar-

geted drug delivery. In a recent study, a stimuli-responsive den-

dronized HA–docetaxel conjugate (HA–DTX–Dendron, HADD)

was synthesized to target delivery of anticancer drug (DTX)

with high biocompatibility into tumor tissues via CD recep-

tors mechanism.[] Similarly, Pan and co-workers reported HA–

doxorubicin nanoparticulate system (with a mean hydrodynamic

size of  nm), interestingly, upon intravenous administration it

showed higher levels of accumulation in the intestines for at least

 h, which has the potential to enhance the selectivity and tar-

getability of the anticancer drugs for colorectal cancer therapy.[]

Anticancer drugs including camptothecin (CPT) have low wa-

ter solubility, and low cellular uptake; hence the use of water-

soluble prodrugs have been investigated to eectively overcome

the low solubility issue of CPT. Xu et al. determined the ef-

fect of HA molecular weight (MW) on the physicochemical

properties, antitumor activity of CPT–HA conjugates in which

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HA acted as an eective solubilization carrier and targeting

molecule.[] In recent years, anticancer drug delivery technolo-

gies have used polymer chemistry and nanotechnology to de-

velop eective dosage forms that address constraints such as

increased targetability, drug cargo internalization, and local on-

site delivery with reduced o-targeted side eects. Zhang et al.

reported a novel multifunctional mesoporous silica nanoparti-

cles (MSN)-based biotin/HAase dual-stimulus-triggered delivery

system for targeted therapeutic delivery of doxorubicin (DOX);

further this study hypothesized that once MSN-HA/Dox is pref-

erentially taken up by cancer cells via receptor-mediated endo-

cytosis, drug release is triggered by HAase-mediated degrada-

tion of HA, which is enhanced further by desthiobiotin displace-

ment by intracellular biotin.[] In a similar study, Sauraj et al.

reported xylan--ﬂuorouracil--acetic acid (Xyl--FUAC) prodrug

conjugate for colon cancer therapy, in which free drug showed

more cytotoxic eects compared to conjugate form in in vitro

studies.[] Taken together, these ﬁndings indicate that, modiﬁed-

HA-drug conjugate systems are eective in tumor-targeting with

increased therapeutic ecacy and stability.

Recently, lot of eorts were kept in developing polymers that

carry combination of dierent therapeutics with improved out-

comes. Krishnan et al. developed clinically translatable polymer–

drug conjugates of doxorubicin and camptothecin called Dox-

orubicin and Camptothecin Tailored at Optimal Ratios (DOC-

TOR) for topical treatment of skin malignancies using HA, where

DOCTOR outperformed the clinical standard (Efudex) in terms

of cancer-cell killing selectivity while being safe for healthy skin

cells.[]

In another recent study, HA-based hydrogel conjugates were

studied for their ability to change the rheological characteris-

tics using supramolecular chemistry as a physical interaction

with cyclodextrin (CD) constructs. The supramolecular system

(between CD and PEG) and catechol polymerization (between

dopamine molecules grafted to the HA polymer) could be used

as dual physical and chemical crosslinking strategies for tunable

hydrogels for long acting donepezil delivery via subcutaneous

administration[] (Figure 5).

In addition to the anticancer drug delivery applications, in-

teractions of HA and CD have been used in investigations

of atherosclerotic plaques.[, ] In a recent study, the hydropho-

bic atorvastatin (ATV) was constructed as the core of the HA

nanoparticle (mean size  nm) system (HA-ATV-NP) capable

of delivering a substantial amount of ATV per particle. When

compared to free drug, HA-ATV NPs can target inﬂammatory

atherosclerotic plaques via the CD receptor and release their

payload with increased therapeutic ecacy.[] Apparently, the

well-known antimalarial medicine hydroxychloroquine (HCQ)–

HA conjugate system was recently demonstrated to improve

bioavailability and treatment eectiveness for COVID- illness

through its versatile drug conjugation technology. The HA–HCQ

conjugate and HCQ were evaluated independently against four

distinct SARS-CoV- target proteins using molecular docking

experiments.[] Despite encouraging results from several in vitro

and in vivo experiments, chemically modiﬁed or HA-conjugated

formulations required more pharmacokinetics and dynamics

understanding before being used in therapeutic settings. Even

though eorts are made in this direction, further studies need

to be established on the degree of substitution of drug moieties

on HA chain/system and its eect on stability, release, pharma-

cokinetics, etc. In Table 1 various recently reported HA-small

molecule drug conjugates are shown.

5. HA-Based Macromolecule Conjugates

Macromolecules (such as protein and peptide drugs) possess

limitations that hinder their clinical potential, due to low wa-

ter solubility, low speciﬁcity, can cause associated o-targeted

side-eects. These agents have challenges in targeting to deliv-

ery sites, and required larger doses due to their short half-lives.

Recently, several drug delivery systems have been investigated as

potential solutions for overcoming these aforementioned short-

comings. In particular, HA has been investigated for its use as a

promising macromolecule drug carrier due to many advantages

including high water-solubility, biocompatibility, and targeting

ability with selective interaction with receptors.[] Additionally,

HA displays a very high loading capacity due to the presence of a

large number of hydroxyl and carboxyl groups on HA as well as its

reducing end that serves as a masked aldehyde to conjugate ther-

apeutics to terminal chain.[] In order to overcome some of the

disadvantages associated with utilizing HA as a drug carrier, vari-

ous chemical modiﬁcations (mentioned in the previous sections)

have been developed, including esteriﬁcation of HA, chemically

modifying HA with carbodiimide, and crosslinking of HA with

either divinyl sulfone, glycidyl ether, or dialdehyde.[] In this sec-

tion, we will discuss and highlight recent advancements in the

HA-based systems for peptide and protein delivery for more e-

cient therapeutic applications (Table 2).

Recently, several works have investigated conjugation of HA

to serum albumin proteins in order to promote improved anti-

cancer drug delivery with enhanced therapeutic ecacy. Albu-

min (an endogenous protein) has been regarded as a promis-

ing drug carrier due to its nontoxicity, biodegradability, and

biocompatibility.[] A study by Edelman. et al. utilized the

HA–CD complex to create HA–bovine serum albumin (BSA)

nanoparticle conjugates loaded with paclitaxel. Researchers ob-

served that Maillard reaction-based covalent conjugates of BSA–

HA were able to self-assemble into nanoparticles (≈tonm)in

which hydrophobic molecules such as paclitaxel could be loaded

eciently. The paclitaxel-loaded BSA–HA nanoparticles were

selectively internalized, displayed high toxicity against CD-

expressing cancer cells against cells lacking CD expression

with selective targeting mechanism.[] This work suggests that

BSA–HA conjugates may be used as eective nanodelivery plat-

forms to enhance the delivery of hydrophobic chemotherapeutic

drugs.

In addition to research being done on anticancer drugs conju-

gated with HA to utilize HA’s binding anity for CD+cells,

HA derivatives are being investigated as potential facilitators for

prolonging the therapeutic activity of certain anti-inﬂammatory

medications. Over the last two decades, there have been multi-

ple studies in which researchers have investigated the improved

therapeutic eects of synthesizing conjugates of crosslinked-HA

with anti-inﬂammatory medications such as dexamethasone to

treat postoperative peritoneal adhesions,[] oligoethyleneimine

to treat ulcerative colitis[] (Figure 6), and anti-TNF-𝛼to help

heal severe burns.[] HA has drawn particular attention in the

ﬁeld of arthritic diseases because, in addition to improving clear-

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Figure 5. a,b) Schematic synthesis illustration of polypseudorotaxane and polydopamine-based hydrogel structures, as well as the design and fabrication

of a PDM-loaded HD-PEG/-CD hydrogel construct for subcutaneous, long-acting injectable delivery of donepezil. Reproduced with permission.[46]

ance time and stability of antiarthritic drugs, HA is also a major

component of the cartilage extracellular matrix and can poten-

tially provide increased therapeutic ecacy when conjugated to

anti-inﬂammatory agents.[]

A relatively new ﬁeld of research, progressively more stud-

ies are being conducted on the eects of anti-neurodegenerative

drugs conjugated with HA. HA has been considered an ideal

candidate as both a central nervous system tissue engineering

scaold and drug delivery device with regards to biocompatibil-

ity due to the existence of HA in the extracellular matrix of the

brain.[] HA has also been suggested for use alongside mes-

enchymal stem cell therapy as a tool to combat neurodegenerative

disease. Mesenchymal stem cells are currently being researched

as a potential method of regenerating neural networks in inﬂam-

matory sites where neural injuries or degeneration occur and be-

cause they express CD, it is proposed that HA’s anity for

CD might lead to some novel targeted anti-neurodegenerative

therapies.[]

Similarly, engineered hydrogels based on HA and ﬁbronectin

were examined for their mechanobiological characteristics on hu-

man mesenchymal stem cells (MSCs), which mimic the quali-

ties of ECM to govern stem cells in both D and D settings.[]

Moreover, studies on HA-based antibody conjugates conﬁrmed

the dual targeting ability of therapeutic agents in liver metas-

tasis treatment. Lee et al. reported a novel hyaluronate (HA)–

death receptor  antibody (DR Ab) conjugate system by chem-

ical conjugation between the hydrazide group of PDPH ((-(-

pyridyldithio)propionyl hydrazide) and aldehyde group of DR

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Tabl e 1 . Examples of recent reports on HA-small molecule drug conjugates, chemistry, and therapeutic applications.

Bioconjugation type Therapeutic agent Description of the conjugate and

linkage

Therapeutic application and outcome Refs.

Conjugation of doxorubicin

(DOX) and camptothecin

CPT to HA

Doxorubicin and

Camptothecin

Dual drug(s) covalent conjugated HA

micelles (amide and ester linkages)

•Skin cancer lesions

•Enhanced cancer-cell killing speciﬁcity with

superior safety to healthy skin cells)

[45]

HA–docetaxel conjugate

(HA–DTX–Dendron, HADD)

Docetaxel Conjugation of mal-GFLG-DTX,

mal-Cy5.5, and Dendron-8-Glu to HA

•Triple negative breast cancer (TNBC)

•HA in HADD increased DTX delivery to

tumor cells with rich CD44 receptors

interaction.

•HADD inhibited tumor growth in

MDA-MB-231 tumor-bearing mice by

99.71%, compared to free DTX

[40]

HA–PEG/𝛼-CD hydrogel system Donepezil Polypseudorotaxane structure,

polydopamine bond-based

crosslinked HA hydrogel

•The HD-PEG/𝛼-CD/PDM 8.5 injectable

hydrogel system gelled immediately and

delivered donepezil in sustained manner

via subcutaneous route

[46]

HA–hydroxychloroquine

conjugate (HA–HCQ)

Hydroxy chloroquine Conjugation of HA–HCQ via ester

linkage

•In silico antiviral activity of HA–HCQ

toward diﬀerent SARS-CoV-2 protein

molecular targets

[49]

HA–metformin (MET)

phospholipid sonocomplexes

(HA–MPS)

Metformin Conjugation of HA–MET via amide

linkage

•Pancreatic ductal adenocarcinoma

•Enhanced lipophilicity and targeting

potential of MET via CD44 binding

[50]

HA–doxorubicin conjugate

(HA–DOX)

Doxorubicin HA–DOX conjugates via nucleophilic

acyl substitution

•Colorectal cancer

•HA–Dox shows higher accumulation in the

intestinal regions upon intravenous

injection with enhanced inhibition of

colorectal cancer

[41]

HA-conjugated hybrid

nanoparticle system

Curcumin HA surface-conjugated PLGA–chitosan

nanoparticle (amide linkage)

•Colon targeted drug delivery

•HA functionalized nanohybrid particles are

eﬀective in delivering loaded drugs orally

to the lower gastrointestinal tract (GIT)

[51]

HA–carnosine conjugates

(HyCar)

Carnosine Linking of HA to Car through an amide

bond

•Neurodegenerative disorders (Alzheimer’s)

•HyCar inhibits the formation of

amyloidtype aggregates and able to

dissolve the amyloid ﬁbrils and to reduce

A𝛽-induced toxicity

[52]

HA–paclitaxel conjugate

(HA–PTX)

Paclitaxel Self-assembled HA–PTX polymeric

prodrug and methoxy poly(ethylene

glycol)–poly (lactide) block

copolymer (mPEG-PLA) to produce

nanoparticle composite (mPPHP

NPs)

•Antitumor activity

•mPPHP NPs preferentially accumulate in

the tumor site via EPR eﬀect and enter

cancer cells by CD44-mediated endocytosis

[53]

HA–atorvastatin (ATV)

conjugate

Atorvastatin Conjugation of ATV-amine 7 with

carboxylic acid of HA via amide

linkage

•Inhibition of atherosclerotic plaque

inﬂammation

•Potential of delivering ATV, with CD44

receptor targetability in inﬂammatory

atherosclerotic plaques

[48]

PMX-conjugated HA

(HA-ADH-PMX)

Pemetrexed (PMX) HA-ADH-PMX synthesized by reacting

HA-ADH with

N-hydroxysuccinimide-activated

PMX (NHS-PMX)

•For malignant pleural mesothelioma

(MPM) treatment

[54]

HA–QT conjugate micelles Quercetin Conjugates of HA–quercetin (HA–QT)

via ester linkage

•A tumor cell-targeted prodrug was

developed for QT, using HA conjugate

micelle carrier

•Shown enhanced inhibition on tumor

growth in H22 tumor-bearing mice

[55]

(Continued)

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Tabl e 1 . (Continued).

Bioconjugation type Therapeutic agent Description of the conjugate and

linkage

Therapeutic application and outcome Refs.

HA-SS-DOX prodrug

self-assembled nanoparticles

Doxorubicin Hydrophilic HA-SS-N3 with a

hydrophobic DOX conjugate

copolymer self-assembled

nanoparticle

•Targeted cancer chemotherapy

•NPs were eﬀectively internalized by tumor

cells overexpressed by the CD44 receptors

[56]

Cur–HA–SPu conjugate Curcumin Curcumin grafted HA modiﬁed pullulan

polymers by esteriﬁcation

•Cur–HA–SPu polymer showed accelerated

wound healing ability with anti-microbial,

antioxidant activities

[57]

HA–MTX conjugate Methotrexate HA–MTX conjugate with the ester

linkage

•Rheumatoid arthritis (RA)

•Ester linkage cleavable in a mildly acidic

environment of RA

•HA–MTX conjugate has promising

potential for RA therapy

[58]

HA–cinnamaldehyde (CA)

nanoﬁbers

Cinnamaldehyde CA conjugation to HA through

acid–labile hydrazone bond

•Antitumor eﬀect by economized

photodynamic therapy

•Novel strategy that speciﬁcally boost tumor

oxidative stress for reinforced oxidation

therapy

[59]

HA–icariin (HA–Ica) conjugate

hydrogel

Icariin Methacrylic anhydride (MA)-modiﬁed

icariin (MIca) nanoparticles

dispersed in HA–MA hydrogel

•HA–Ica hydrogel exhibited controlled

release of Ica with good cytocompatibilty.

•Potentially used as a scaﬀold for cartilage

tissue engineering

[60]

Chol–Suc–HA conjugate

self-assembled nanoparticles

(NPs)

Docetaxel (DTX) Amphiphilic cholesteryl succinoyl HA

(Chol–Suc–HA) conjugates

self-assembled into DTX-loaded NPs

(ester linkage)

•Chol–Suc–HA NPs were promising drug

vehicles for systemic targeting of

CD44-overexpressed cancers

[61]

Ab. Further, pyridyldisulﬁde group of PDPH–DRAb conjugated

to thiol group of  kDa thiol end-modiﬁed HA. Interestingly, due

to the dual-targeting ability, HA–DR Ab conjugate seemed to be

highly accumulated in the liver and have shown eective inhi-

bition of tumor growth in liver metastasis mice model (Figure

7). Despite the advancements of HA-based macromolecular con-

jugates; molecular weight dependent eects, speciﬁc conjugates

characterization, puriﬁcation and conjugates kinetic, mechanis-

tic aspects should be studied for further in order to enter into

clinics.

6. Combination of HA and Other Biopolymer

Crosslinked Systems

Growing evidences demonstrate that biopolymer scaolds can

be formulated by blending or crosslinking HA with naturally

derived or synthetic macromolecules for various biomedical

applications.[, ] Recent ﬁndings suggest that intra-articular

injections of nonchemically modiﬁed hybrid complex of high

molecular weight (HMW) sodium hyaluronate and sodium chon-

droitin without crosslinking agents has eectively reduced hip

pain and osteoarthritis functionality over time in symptomatic

hip osteoarthritis patients.[] The polymer interactions such as

hydrophobic and electrostatic interactions allow the combination

of the HA and other biopolymers such as collagen to form newly

blend matrices.

Interestingly, in another study, an optimal self-assembled bio-

component hydrogel of poly(ethyleneglycol) (PEG), crosslinked

type I/II collagen combined with HA has been demonstrated

to mimic the D inﬂammatory microenvironment of the in-

tervertebral disc that can regulate cell shape, extracellular ma-

trix content, and inﬂammatory signaling with understanding the

role of glycans.[] Biofunctionalization of collagen and high-

sulfated HA on the biodegradable synthetic scaold of three-

armed methacrylate-terminated macromers/PEG increased scaf-

fold pore size that has maintained cell viability, improved osteo-

genesis, and mineralization of new bone matrix.[] Besides, HA

has been also combined with laminin–hydrogel as a luminal ﬁller

for collagen-based and chitosan-based nerve conduits that have

shown functional recovery and tissue regeneration in the sciatic

nerve injury model.[] The combination of HA with sodium al-

ginate (as bioink system, capable of being printed without em-

ploying chemical crosslinking agents) exhibited higher viscos-

ity, by maintaining the gross structure of the total hydrogel,[]

here HA increased the bioactivity of the encapsulated cells. Simi-

larly, the D-printed chitosan scaold, when combined with HA,

has shown a higher mechanical strength while maintaining its

structure, allowing chondrocytes to inﬁltrate into the scaold and

colonize the pores, forming ﬁbroblast-like cells and promoting

chondrogenesis.[]

It is well known that chemical modiﬁcation of functional

groups of HA will allow covalent crosslinking of the biopoly-

mer. The carboxylic group (–COOH) can be modiﬁed through

the process of amidation, Ugi condensation, and ester forma-

tion, whereas the hydroxyl group (–OH) of HA can be modiﬁed

via the ether, hemiacetal formation using glutaraldehyde; ester,

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Tabl e 2 . Recent ﬁndings of HA–macromolecular conjugates to facilitate enhanced targeted delivery, and/or therapeutic eﬀects.

Macromolecule type Description of conjugation and design Therapeutic application Research outcome Refs.

Interferon-𝛼(INF𝛼) Aldehyde-modiﬁed 100 kDa HA

dissolved in 5 mg mL−1sodium

acetate buﬀer and conjugated with

IFN𝛼

Hepatitis C virus (HPC) •Rate of intracellular uptake was

signiﬁcantly higher for HA–INF𝛼system

•HA–INF𝛼system showed signiﬁcantly

enhanced targeting of liver and longer

residence time

[65]

Bovine serum albumin

(BSA)

BSA and 6.4 kDa HA conjugate with

paclitaxel

Targeted cancer therapy •Improved selective internalization and

increased toxicity on CD44+cells for

BSA–HA system compared to HA and BSA

alone

[66]

Human serum albumin

(HSA)

HSA and erlotinib (ERT) NPs modiﬁed

with 5400 Da HA

Lung cancer •ERT albumin nanoparticles modiﬁed with

HA/HSA showed superior antiproliferative

eﬀect on lung carcinoma cells compared to

free ERT and ERT–HSA nanoparticles

[67]

Glucagon Microneedle array patch of

methacrylated-HA, combined with

insulin formed via polymerization

with crosslinker/photoinitiator post

UV irradiation

Hypoglycemia •Microneedles were able to release

glucagon in response to elevated insulin

levels in interstitial ﬂuid in vascular and

lymph capillary networks, preventing the

risk of hypoglycemia

[68]

Branched

oligoethyleneimine (bOEI)

bOEI-100 kDa HA conjugate system

synthesized via condensation

reaction

Ulcerative colitis (UC) •Intraperitoneal injection of bOEI–HA in

UC-induced rats showed signiﬁcantly less

inﬂammatory activity via less secretion of

TNF-𝛼, IL-6, and PGE2 compared to bOEI

alone

[69]

Epidermal growth factor

(EGF)

HA–EGF conjugate synthesized via

coupling reaction between

aldehyde-modiﬁed HA and

N-termine amine group in EGF

Skin wound healing and

regeneration

•More eﬃcient skin regeneration and

transdermal delivery from HA–EGF patch

into normal skin and peripheral tissues

near wound site than free EGF

[70]

Bilirubin (BR) 100 kDa HA–BR systems to facilitate

the formation of self-assembled

nanoparticles

Ulcerative colitis (UC) •HA–BR system showed signiﬁcantly

increased expression of anti-inﬂammatory

cytokines and decrease in proinﬂammatory

cytokines.

•Improved microbiota diversity and eﬀective

targeting via CD44 on the gastrointestinal

endothelium

[71]

O-aminophenol HA and o-aminophenol conjugated via

condensing reaction with carboxyl

groups of HA

Matrix metalloproteinase 2

(MMP-2) inhibition

•Signiﬁcant inhibition of MMP-2 expression

with HA-o-aminophenol conjugate

compared to HA conjugates with other

biologically active amines including

Procaine and Streptocide

[72]

Interferon alpha (INF𝛼) 2a Acetyl-modiﬁed HA conjugated to

INF𝛼2a via an aldehyde-quenching

reaction

Ovarian cancer •HA–INF𝛼2a conjugate showed an

improved anticancer eﬃcacy, signiﬁcantly

enhanced survival compared to free

INF𝛼2a

[73]

Death receptor 5 antibody

(DR5 Ab)

Chemical conjugation pyridyldisulﬁde

group of PDPH–DR5Ab conjugated

to thiol group of 5 kDa thiol

end-modiﬁed HA

Liver metastasis •In vivo bioimaging showed that HA–DR5

Ab conjugate highly accumulates in the

liver and is signiﬁcantly more eﬀective in

inhibiting tumor growth compared to DR5

Ab alone

[74]

Carnosine Carboxyl group on 200 and 700 kDa

HA conjugated with N-terminal

group of carnosine

Alzheimer’s disease (AD) •HA–carnosine conjugate inhibits the

formation of A𝛽aggregates, dissolves

amyloid ﬁbers, and reduces A𝛽-induced

toxicity in vitro compared to free carnosine

and HA alone

[52]

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Figure 6. a) Schematic illustration of the branched oligoethyleneimine (bOEI) conjugate system synthesized via condensation reaction; conjugate physic-

ochemical properties such as b,c) bOEI-HA gel volume, d) surface charge/zeta potential, and e) N-acetylglucosamine release comparison between native

HA to bOEI-HA. Further, complete biological studies revealed that the conjugated form eﬀectively modulated the inﬂammatory response, enhanced the

carbamate formation, and oxidization with sodium periodate.[]

For example, tyramine-modiﬁed HA synthesis through oxidation

has been used for in situ crosslinking systems, employing hydro-

gen peroxide (HO) and horseradish peroxidase (HRP) to initi-

ate crosslinking reaction at the position C–CandC

–O between

phenols. When combined with a porous D silk ﬁbroin matrix,

it can form a tunable hybrid scaold, which revealed the higher

chondroinductive and biomechanical properties tailored for car-

tilage repair.[] Amidation with carbodiimides is a common strat-

egy for HA modiﬁcation in the carboxylic group. The initial step

in the reaction is the activation of the carboxylic acid of HA by

EDC, which results in the formation of an O-acyl isourea inter-

mediate. The nucleophilic attack of the amine on the activated

HA in the second step of the reaction results in the formation

of the amide bond.[] For example, activation of carboxyl groups

of HA through carbodiimide mediated-reaction using EDC cou-

pling with N-hydroxysuccinimide (NHS) to stabilize the inter-

mediate, results in the formation of the amide bond in the HA

structure that can react with succinimidyl groups of -arm PEG

amine to form crosslinked PEG/HA-based hydrogels.[] Vario us

crosslinked HA-hybrid systems are compared and enumerated

in Table 3.

Besides, PEG/HA hydrogel has shown a therapeutic eect

in preventing pain, speciﬁcally alleviating thermal hyperalge-

sia and mechanical allodynia, downregulating spinal nocicep-

tion markers, and hyperinnervation, altering glycosylation, and

modulate inﬂammatory and protein regulatory pathways for in-

tervertebral disc repair in vivo (Figure 8).[] In various studies,

-(,-dimethoxy-,,-triazin--yl)--methylmorpholinium chlo-

ride (DMTMM) was also widely used as a crosslinking initiator

in addition to EDC. The biphasic dispersion of crosslinked HA

in non-crosslinked HA solution was formulated using DMTMM

to initiate crosslinking reaction to form crosslinked PEG/HA that

exhibited a dendritic needle-like shape morphology, biphasic-HA

system decreased inﬂammation and permeability in an in vitro

interstitial cystitis model.[]

It is also well known that Schi base reaction is a mechanism

of the crosslinking system of HA that reacts between aldehyde

groups of modiﬁed-HA and amino groups of other polymers

to form a hydrogel. For example, the combination of HA and

gelatin hydrogel has been used to modify surface hydrophilic-

ity of implantable polydimethylsiloxane (PDMS) via the Schi-

base reaction of the aldehyde of HA (CHO-HA) and amine of

gelatin (Gel-NH) using EDC/NHS, thus forming the implant

that has higher biocompatibility and shown to improve extra-

cellular matrix deposition, and attenuate capsule formation and

inﬂammation in vivo.[] The crosslinking amino group of O-

carboxymethyl chitosan and aldehyde HA via the Schi-based re-

action forming the porous structure of the chitosan/HA hydro-

gel, the reported gel has the capability of promoting abdominal

endothelial regeneration.[] Dual-crosslinking systems consist-

ing of noncovalent and covalent binding have provided an e-

cient strategy to ﬁne-tune hydrogel injectability, degradation, and

mechanical properties. For example, electrostatic interactions be-

tween cationic methacrylate chitosan and anionic HA allowed

Schi-based reaction to enable rapid gelation that can be used as

a cell delivery system and D printing of encapsulated cell scaf-

folds. The mechanical properties of the hydrogel network can

be improved through covalent crosslinking using in situ pho-

topolymerization of methacrylates.[] Various recent key HA-

based conjugate systems, and their potential interactions, diverse

biological responses are enumerated in Table 4.

HA has been regarded as a potential therapeutic mediator

for inﬂammatory bowel disease (IBD) in part due to its large

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Figure 7. a) Schematic illustration of dual-targeting mechanism of HA–DR5 Ab conjugate by targeting DR5 and HA receptors and synthesis scheme. b)

In vivo bioimaging of the liver studies revealed that enhanced ﬂuorescence signal was found with HA–DR5 Ab conjugate, compared to free or mixture.

c) The in vivo bioimaging and antitumor eﬃcacy of HA–DR5 Ab conjugate in liver metastasis mice model Reproduced with permission.[74] Copyright

2016, American Chemical Society.

presence in the extracellular matrix of the gastrointestinal tract

(GIT) and similarity to mucins lining the GIT, suggesting that it

plays a role in increasing epithelial barrier integrity.[] In addi-

tion, HA’s targeting ability may also be exploited in IBD as the

CD receptor is expressed on endothelial cells in the GIT.[ ]

In one of the recent study, researchers formulated HA–

bilirubin (HA–BR) conjugated nanoparticles (mean size  ±

 to  ± nm) for the targeted treatment of IBD, in which

researchers were able to facilitate the aqueous formulation of

BR and exploit HA–CD interactions to target inﬂamed colon

epithelium and proinﬂammatory macrophages via oral delivery

of the HA–BR nanomedicine system. The HA–BN system has

shown a potent reactive oxygen species (ROS)-scavenging eect

in protecting colonic epithelium against ROS-mediated cytotoxic-

ity, resistance to hyaluronidase-mediated degradation, modulated

gut microbiota, recovered tight junction proteins, reduced apop-

tosis, permitted intestinal barrier functions, and exhibited anti-

inﬂammatory eect in an acute colitis model of mice[] (Figure

9). In a similar study, Kotla et al. reported the development of an

enema suspension system based on hyaluronan (HA) that pro-

tects the dysregulated colon epithelium by decreasing inﬂamma-

tion, permeability, and retaining the epithelial barrier integrity

in dextran sodium’s sulphate (DSS)-induced colitis mice model

with proteomic proﬁling analysis.[]

7. HA Derivatives as Cell-Based Therapeutics

The biological signiﬁcance of HA, speciﬁcally its recognition ca-

pacity to alter cell behavior, tissue mechanics, and regeneration

capabilities, paves the way for the development of biomateri-

als incorporating HA for cell-based systems. These scaolds has

unique therapeutic properties due to the nature of HA molecules

capable of interacting with proteins, including aggrecan, versi-

can, lymphatic vessel endothelial receptor  (LYVE-), and tu-

mor necrosis factor-𝛼stimulated gene  (TSG-), and mem-

brane receptors, including a CD, hyaluronectin, hyaluronan-

mediated motility (RHAMM), stretch-activated channels,[ ] and

Ca+channels.[ ] By the aforesaid interactions, HA plays a key

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Tabl e 3 . The therapeutic applications of HA with other combinatorial biopolymer systems.

Polymer scaﬀold HA modiﬁcation and crosslinking system Findings Refs.

Hybrid complex of sodium

hyaluronate and sodium

chondroitin

Blending of hyaluronate 2.4% and sodium

chondroitin 1.6%

•Intra-articular administration was tolerated in patients

•Eﬃcacy of treatment to reduce visual analogue score of

hip pain

•Eﬃcacy of treatment to reduce osteoarthritis functionality

[84]

Type I and II collagen/HA hydrogels 4-arm-PEG-SG crosslinked collagen

blended with HA

•Maintained the cell morphology of nucleus pulposus and

annulus ﬁbrosus

•Decrease extracellular matrix expression of type II

collagen and aggrecan

•Regulate inﬂammation of IL-6 and TNF-𝛼, and suppressor

of cytokine signaling proteins (SOCS) signaling

[85]

Biofunctionalization of collagen and

high-sulfated HA on three-armed

methacrylate-terminated

macromers/PEG

Collagen and HA coating on freeze-dried

scaﬀold

•Increased eﬃciency of coating with collagen and

sulphated HA on high pore scaﬀold

•Coating stability over 7 days

•Elevated of ALP for osteogenic marker

•Histologically evidence of mineralized matrix

[86]

HA–laminin–hydrogel ﬁller in

collagen-based and chitosan-based

nerve conduit

Blending of HA and laminin •Functional motor recovery in compound muscle action

potential (CMAP) of the tibialis anterior

•Reinnervation improve muscle mass–muscle weight

ratios of hind limb muscles

•Reconstruction of sciatic nerve indicated by toluidine blue

staining. Regeneration of Schwann cells cultured in

hydrogel

[87]

Bioink of HA and sodium alginate

(SA) hydrogel

Blending of HA and sodium alginate for

3D bioprinting system

•Viscosity of bioink increased with HA

•HA enhances the bioactivity in the encapsulated cells

•Printed hydrogel maintained its gross structure and

successfully printed layer-by-layer hydrogel upon addition

of CaCl2.

[88]

3D-printed chitosan/HA scaﬀold Blending of HA and chitosan for 3D

bioprinting system

•Maintained the structure of scaﬀolds for up to 6 months

in water and 21 days in culture media

•Chondrocytes penetrated scaﬀold, colonized the pores of

the scaﬀold

•Increased expression of chondrogenic markers (collagen

type II, aggrecan and versican, SOX9 and RUNX2)

[89]

Silk ﬁbroin matrix

and a HA hydrogel

Tyramine-modiﬁed HA crosslinked with

H2O2and HRP concentrations blended

in silk ﬁbroin matrix

•Increase in storage modulus (G′) (elasticity), high viability

on human articular chondrocytes, upregulation of

COL2A1 and SOX9 expression

•Surface morphology of heterogenous and macroporous

3D scaﬀold and ﬁbroblastic-shape chondrocytes

•Higher sulphated glycosaminoglycan content

[90]

Crosslinked PEG/HA hydrogel Carboxyl groups of HA with primary

amines of PEG to form the crosslinked

hydrogel (carbodiimide chemistry)

•Hydrogel inhibited hyperinnervation of sensory nerve

ﬁbers (GAP43), neuropeptides (CGRP), pronociceptor

(TrkA and TRPV1)

•Hydrogel suppressed spinal nociception markers (c-Fos

and substance P), altered glycosylation and modulated

inﬂammatory signaling of IL-6 and IL-1𝛽.

•Hydrogel regulated SMAD3/TGF-𝛽3 for matrix remodeling

[92]

Biphasic dispersion of crosslinked

HA in non-crosslinked HA solution

DMTMM initiated crosslinking of HA and

4-arm PEG

•Maintained cell metabolic activity, decreased IL-8 level in

HTB-2 cells study

•Decreased the apparent permeability of T84 cells

[93]

HA/gelatin hydrogel on PDMS

implant

Surface coating of hydrogel on PDMS

through Schiﬀ-base reaction employing

aldehyde of HA (CHO-HA) and amine of

gelatin (Gel-NH2) using EDC/NHS

•H-NMR of CHO–HA and gel–NH2spectra to inform

surface conjugation of PDMS and gelatin

•Higher viability of human adipose-derived stem cells

•Loose collagen ﬁbers distribution, downregulation of

COL1A1, COL1A3, TGF-𝛽1, 𝛼-SMA, SMAD3, IL-𝛽1, and

IL-6

[94]

(Continued)

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Tabl e 3 . (Continued).

Polymer scaﬀold HA modiﬁcation and crosslinking system Findings Refs.

Chitosan/HA hydrogel Crosslinking amino group of

O-carboxymethyl chitosan and Aldehyde

HA via the Schiﬀ-based reaction

•Maintained viability of ﬁbroblasts encapsulated in the

hydrogels with round shaped and uniformly distribution

•Increase thickness of the regenerative endothelial tissue

•Positive stained of CD31 and 𝛼-SMA for mature vascular

formation, positive stained of vimentin and COL1A2 for

extracellular matrix (ECM)

[95]

Dual-crosslinked chitosan

(CHMA)/HA (OHA) hydrogel

Methacrylate chitosan and aldehyde

functionalized HA via dynamic

electrostatic and Schiﬀ-based reactions,

and in situ covalent chemistry using

photopolymerization of methacrylates

•Steady moduli for crosslinked hydrogel under the time

sweep rheology, higher moduli for photopolymerization

hydrogel

•Dynamic crosslinked hydrogel with reversable behavior

gel-liquid phase under the shear stress

•Rat bone marrow derived stem cells encapsulated in

hydrogels had higher viability and homogenous cell

distribution

[96]

Figure 8. a). Schematic representation of crosslinked high-molecular weight HA and four-arm PEG amine hydrogel composite. b) Implantation of HA

hydrogel in the injured intravertebral disc region. c) Mitigation of the injury-induced pain phenotype by the implanted HA hydrogel, confocal schematic

representative staining demonstrated the presence in AF and NP tissues, particularly in the untreated injury group, of the GAP43 protein (yellow label)

Association for the Advancement of Science (AAAS).

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Tabl e 4 . Various HA-based conjugate systems, potential interactions, and diverse biological responses.

HA system HA interactions and model applications Other ﬁndings Refs.

Acellular PEG/HA hydrogel Mechanism through HA–CD44 interaction

Model – In vitro inﬂammation model of

nucleus pulposus (NP)

•Hydrogel stability for up to one month in PBS

•Noncytotoxic in nucleus pulposus cells

•Attenuated inﬂammation, downregulated neurotrophic factor

[91]

DSPE–HA conjugates on

Gli1 siRNA nanoparticles

DSPE–HA binding aﬃnity to CD44 receptor

via

Endocytosis

Model – In vivo relapsed tumor models of

gastric CSCs

•Successful conjugation of DSPE and HE indicated by spectrum

of methylene groups with a sharp absorbance peak of carbonyl

in the acyl chains of DSPE and peak of hydroxyl groups of HA

•Increased of mean diameter of Gli1 siRNA nanoparticles

•Cellular uptake of CD44+gastric cancer stem cells of siRNA

nanoparticles. Inhibited cellular proliferation of CD44+CSCs

[97]

Mesenchymal stem cell

extracellular vesicles with

HMW–HA

MSC EV bound to HMW HA results in the

interaction of CD44 receptor on

mesenchymal stem cell extracellular

vesicles (MSC EV)

Model – Pseudomonas aeruginosa

(PA)-induced pneumonia in mice model

•Increased MSC EV with HA uptake by human monocytes

•Increased bacterial phagocytosis by LPS stimulated human

monocytes

•MSC EV with CD44 siRNA treatment reduced bacterial

phagocytosis

•Intravenous administration of MSC EV with HA-reduced

Pseudomonas aeruginosa CFU levels

[98]

HA–bilirubin

nanomedicine system

HA–CD44 interactions.

Model – Ulcerative colitis mice model

•HABN exhibited ROS-scavenging activity and protected

colonic epithelial cells from ROS-mediated cytotoxicity

•HABN protected and recovered DSS-induced colitis phenotype

and improved intestinal barrier function of tight junction

associated proteins (ZO-1 and occluding-1), induced

expression of anti-microbial peptides (murine-𝛽defensin) and

regulated colonic microbiota

•HABN reduced colonic epithelium apoptosis, attenuated

inﬂammation (IL-1𝛽,TNF-𝛼, IL-6), monocytes, and

neutrophils; increased IL-10 and TGF-𝛽

[71]

HA solution Interaction of HA and TRPV1 channels via

the hydrogel bonding on the positively

charged amino acid of TRPV1

Model – Rats and TRPV1 null mice model

•Reduced capsaicin evoked TRPV1 membrane ionic currents in

HEK-TRPV1- EYFP (+) cells; reduced TRPV1-single-channel

activity in HEK-TRPV1-EYFP (+) cells

•Reduced latency of pain responses to heat following

subcutaneous injection of HA in the rat paw, and TRPV1 null

mice

•Reduced capsaicin-evoked nerve impulse activity in saphenous

nerve of knee joint

•Molecular modeling informed positively charged amino acids

(His614, Lys615, and Arg617) were the putative HA binding in

TRPV1 through H-bond with HA

ref. [99]

An injectable HA-based

microrods

Cell adhesion of ﬁbroblasts on HA,

evidence of focal adhesion protein,

paxillin

Model – Myocardial infarction of rats

•Increased adhesion and spreading of neonatal rat ventricular

ﬁbroblast on HA microrods. HA system decreased ﬁbrosis

markers; decreased myoblast phenotype (COL1A2, alpha

smooth muscle actin (𝛼-SMA) through TGF-𝛽1/SMAD3

pathway.

•HA system decreased matrix metalloproteinases (MMPs) of

MMP2 and MMP9 to prevent ECM breakdown

•Increased thickness of left ventricular wall, deposition of loose

collagen ﬁbers, stroke volume, ejection fraction in rats

•Evidence of focal adhesion (actin, paxillin) at the edges of HA

microrods; positive stained of vimentin for ﬁbroblasts and

CD68 for macrophages

[100]

HA solution Activation of TGF-𝛽/BMP signaling

Model – In vitro study of human amniotic

epithelial cells (hAECs)

•Increased proliferation of hAECs, maintained expression of

epithelial cell marker cytokeratin19 (CK19), CD29, CD73,

•and CD166

•Promoted stem cell pluripotent markers of Oct-4 and Nanog

•Increased TGF-𝛽/BMP signaling

[101]

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Figure 9. The schematic representation of hyaluronan–bilirubin nanoconjugate (HABN) role in modulating ulcerative colitis. Synthesized HABN system

showed to be nontoxic and localizes in inﬂamed regions of the colon of DSS-treated colitis mice and exerts therapeutic eﬀects against acute colitis.

role in modulating biological processes such as cell survival, cell

migration, dierentiation, proliferation, and morphogenesis.[ ]

We have previously shown the therapeutic eect of crosslinked

PEG/HA hydrogel to attenuate inﬂammation and expression of

neurotrophic factors in an inﬂammation model of nucleus pul-

posus cells, possibly through binding to cell surface receptor,

CD[] and also in modulating the gut barrier integrity in colitis

condition.[]

Conjugation of di-stearoyl-phosphatidyl-ethanolamine-

hyaluronic-acid (DSPE-HA) on the small interfering RNA

(siRNA) nanoparticles has increased anity to CD, which is

capable of inhibiting glioma-associated oncogene homolog 

(Gli) protein expression and gastric cancer stem cells (CSC)

tumor spheroid and colony formation, and suppress cell migra-

tion, invasion, and evidence of antitumor activity in vivo.[] The

interaction of the CD receptor on mesenchymal stem cell ex-

tracellular vesicles (MSC EV) was associated with antimicrobial

activity, demonstrating an increased binding of MSC EV bound

to HMW HA led to higher MSC EV uptake and bacterial phago-

cytosis, attenuated inﬂammation, and decreased the bacterial

load in Pseudomonas aeruginosa-induced pneumonia in mice.[]

The HA molecule plays a vital role in regulating cell adhe-

sion for cell–cell communications and modulation of diverse bi-

ological functions and matrix homeostasis. Injectable HA-based

microrods have been shown to increase cell adhesion and to

spread rat ventricular ﬁbroblast on HA microrods with evidence

of focal adhesion protein (paxillin and actin) that contributed to

mechanotransduction pathways, led to higher cell proliferation,

decrease expression of myoﬁbroblast phenotype through TGF-𝛽

and SMAD pathway in vitro. Upon injecting HA microrods in a

rat model of myocardial infarction, focal adhesion (actin and pax-

illin), and positively stained vimentin were shown, with increased

ventricular wall thickness and deposition of loose collagen, thus

improving cardiac function including stroke volume and ejection

fraction in rats.[ ] By the activation of TGF-𝛽/BMP signaling,

the HA has been reported to promote proliferation of human

amniotic epithelial cells (hAECs), maintain epithelial markers,

increase levels of anti-inﬂammatory factors (IL- and TGF-𝛽),

and stem cell pluripotent markers of Oct and Nanog.[]

The cross-talk between osteopontin (OPN), integrin 𝛼𝜈𝛽, and

HA was demonstrated in the osteoarthritis (OA) model, in which

the expression of HA was regulated by the interaction between

OPN and integrin 𝛼𝜈𝛽.[ ] The HA has been reported to inter-

act with the ion channel of the nociceptor to exhibit an analgesic

eect. The HA solution inhibited heat and capsaicin evoked-

intracellular calcium concentration in TRPV-EYFP channels ex-

pressed in HEK cells and dorsal root ganglia culture and pre-

vented TRPV sensitization by bradykinin, conﬁrming the inter-

action of HA on TRPV channels of sensitized peripheral sen-

sory terminals. In in vivo, HA reduces the sensitivity of noci-

ceptor endings to noxious stimuli transduced by TRPV and de-

creased TRPV-mediated nerve impulse activity of joint nocicep-

tor ﬁbers. The interaction of HA and TRPV was modeled via the

hydrogel bonding of HA to the positively charged amino acids of

TRPV.[] The interaction of the HA molecule and postsynap-

tic L-type Ca+channels has been implicated in modulating hip-

pocampal synaptic plasticity, in which exogenous administration

of HA has been shown to increase Cav. mediated current.[]

The HA-based systems have been widely used as a cell-delivery

system to mimic the D microenvironment of native tissue and

support cell transplantation for tissue regeneration. Treatment

of human mesenchymal stem cells (hMSCs) suspended with

batroxobin/platelet-rich plasma/HA hydrogel in nucleotomized

intervertebral disc organ culture has maintained cell viability, hy-

drogel integration with NP tissue, promoted hMSCs dierenti-

ation toward NP phenotype indicated by upregulation of aggre-

can and cytokeratin- (KRT) gene expression, representing a

promising stem cell delivery for disc regeneration.[ ]

Intra-articular injection of mesenchymal stromal cells with

HA solution has demonstrated an increase in cartilage thick-

ness and deposition of type I collagen in temporomandibular

joint osteoarthritis of the rabbit model.[ ] Jha et al. reported

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in comparison to the other crosslinkers examined, hyaluronic

acid (HyA) hydrogels crosslinked with the MMP-degradable pep-

tide QPQGLAK promote the highest cardiac progenitor cells sur-

vival, proliferation, and endothelial cell dierentiation.[ ] In an-

other study, combination treatment of catechol-functionalized

HA patch and adipose-derived mesenchymal stem cells (ADSCs)

has demonstrated the reduced size of the diabetic wound, in-

creased angiogenesis markers of CD and von Willebrand fac-

tor, and paracrine signaling molecules of VEGF, IGF-, FGF-,

ANG-, PIK, and AKT in diabetic wound mouse models.[ ] In-

corporation of ADSCs on the composite hydrogels of methacry-

lated HA containing fragmented polycaprolactone electrospun

nanoﬁbers (NFs) has demonstrated elongated morphology of

cells, higher cell proliferation and osteogenic dierentiation

with evidence of type I collagen  (COL), alkaline phosphatase

(ALP), and runt-related transcription factor  (RUNX).[ ] Chi-

tosan/HA scaold provided an excellent microenvironment for

supporting human ADSCs dierentiation toward cartilage indi-

cated by higher cell proliferation and viability with cells attached

to scaold depositing new matrix of type II collagen and ag-

grecan, and upregulation of chondrogenic markers of ACAN,

COLA, and SOX.[ ]

8. HA as a Biocoating Polymer and Its Therapeutic

Prospective

HA has been widely employed for the modiﬁcation of the surface

of several delivery systems. In this sense, HA has found particu-

lar interest in the modiﬁcation of nanoparticles thanks to its an-

ity for CD. This fact perfectly complements the passive target-

ing promoted by the enhanced permeability and retention (EPR)

eect associated with nanoentities and could overcome the lim-

itations of the current approaches toward clinical translation. In

a particular example,[ ] latex nanoparticles were surface modi-

ﬁed with ten dierent natural or synthetic polyions via the layer-

by-layer (LbL) approach (Figure 10a). The carboxylated polyions

(i.e., poly(-glutamic acid), poly(-aspartic acid), poly(acrylic acid),

and HA) display an increased anity toward ovarian cancer cell

lines with respect to sulfated counterparts (Figure b). Although

the binding ability of HA-modiﬁed particles was lower than the

one observed for the other carboxylated polyions, they were ac-

cumulated within intracellular compartments, whereas poly(-

glutamic acid)- and poly(-aspartic acid)-modiﬁed nanoparticles

were preferentially observed at the cell membrane (Figure c).

Moreover, as concluded from the in vivo studies, HA allows

deeper penetration of the nanoparticles in cancerous tissues with

respect to other surface-modiﬁed nanoparticles, which can be

ascribed to its antifouling and swelling behavior (Figure d).

These properties have thus been used to improve the ecacy and

delivery of chemotherapeutic drugs (e.g., raltitrexed) and photo-

sensitizers for photodynamic therapy in a variety of in vitro and

in vivo cancer models.[, ]

Modiﬁcation of nanoparticles with HA is thus regularly

adopted as a common strategy to ensure the performance of novel

therapies for cancer. This includes also cooperative approaches

where several treatments are combined to boost their synergis-

tic actions. For example, HA-decorated polymer nanoparticles

loaded with a small drug (i.e., docetaxel) in the core and coated

with a photosensitizers (i.e., anionic porphyrin) ensured ecient

uptake and colocalization of the therapeutic agents within can-

cerous cells (i.e., MDA-MB-), tremendously increasing light-

induced cell death.[ ] Besides, considering that HA is degraded

in the tumor microenvironment because of the increased levels

of hyaluronidase, HA-coated nanoparticles can preferentially de-

liver their payload at the target tissue.[, ] More complex sys-

tems have been reported in the bibliography that use HA to in-

crease the blood circulation time and tumor accumulation of na-

noentities. In a particular approach,[ ] HA was conjugated to

the surface of the nanoparticles via pH-sensitive imine bonds. In

the tumor microenvironment, HA was deshielded from the sur-

face, exposing the chitosan layer underneath and promoting cell

uptake. The ability of HA to speciﬁcally bind CD has also been

used to capture tumor cells, which could be potentially used as a

diagnosis system with increased sensitivity and selectivity.[]

HA is commonly employed together with positively charged

polyions (e.g., chitosan) to coat the surface of other biomate-

rials via the LbL approach or alternative procedures.[ ] The

modiﬁcation of HA with dopamine endows adhesive proper-

ties and facilitates its incorporation on a wide variety of sub-

strates, representing an alternative or complementary method

to the widely employed LbL approach for the modiﬁcation of

surfaces.[ ] In this sense, the surface of polymeric biomate-

rials was coated with polyvinylamine (PVAm) and dopamine-

modiﬁed HA via the LbL approach.[ ] Michael-type addition and

Schi base reaction between amine groups of PVAm and cate-

chol groups of dopamine facilitated the assembly process, such

coating prevented the growth of Escherichia coli and Staphylococ-

cus aureus and suppressed bacterial growth, demonstrating its

potential to create antibacterial and biocompatible coatings. Sim-

ilarly, HA/chitosan multilayer ﬁlms were assembled on silicon

substrates and inhibited the adhesion and growth of Staphylococ-

cus aureus and Pseudomonas aeruginosa.[ ]

Considering the huge impact of implant-related infections,

that represent more than % of hospital acquired infections, the

development of advanced biomaterials that inhibit bacterial adhe-

sion and bioﬁlm formation is highly desirable. Thus, antibacte-

rial drugs are usually combined within multilayer ﬁlms to create

a synergistic eect between these two approaches. For example,

gentamicin sulfate was loaded into polyacrylic acid/chitosan mul-

tilayer ﬁlms, which were subsequently sealed with an external

layer of HA.[ ] The release of the entrapped gentamicin sulfate

was accelerated in the presence of hyaluronidase secreted by bac-

teria, thus allowing a triggered drug release. In a similar exam-

ple, multilayer ﬁlms of montmorillonite and HA were assembled

on several substrates[ ] (Figure 11a). The degradation of HA in

response to hyaluronidase promoted the release of the drug (Fig-

ure b) and resisted the pathogen adhesion through ﬁlm peel-

ing (Figure c). Consequently, bioﬁlm formation was inhibited,

thus reducing immune response and bacterial infections in an in

vivo model.

Together with bacterial infections, the integration of implants

with the surrounding tissues represents one of the main clin-

ical problems associated with biomaterials. When the integra-

tion with the surrounding bone (i.e., osseointegration) is incom-

plete, micromotions occur at the interface between the bone

and the implant, which ﬁnally results in implant failure. Here

again, the use of HA has played a pivotal role. Hybrid (i.e.,

organic/inorganic) multilayer ﬁlms made from HA, chitosan,

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Figure 10. a) Schematic representation of the layer-by-layer modiﬁcation of latex nanoparticles with carboxylated and sulfated polyions. b) Fluorescence

intensity of ovarian cancer cells incubated with diﬀerent conﬁguration of nanoparticles, indicating that carboxylated nanoparticles have more aﬃnity

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and bioactive glass nanoparticles were grown on glass substrates

and imparted bioactivity to the otherwise bioinert support, as

demonstrated by the formation of apatite-like structures on their

surfaces.[ ] Similar coatings composed of dopamine-modiﬁed

HA and chitosan improved the adhesion and proliferation of os-

teoblasts with respect to the pristine titanium alloy used as a

control.[ ] To further promote osteoblast adhesion and prolif-

eration, HA/chitosan multilayer ﬁlms were decorated with RGD

peptide (i.e., arginine–glycine–aspartic acid) via carbodiimide

chemistry or formation of disulﬁde bonds.[, ]

Moreover, considering the intrinsic antibacterial properties

of HA/chitosan multilayer ﬁlms discussed above, this strategy

could solve two of the main problems encountered in orthopaedic

implants, namely, bacterial infections and poor osseointegration.

Despite the adhesion of osteoblasts in vitro seems to be poorer

in HA-modiﬁed implants than in pristine counterparts,[ ] in

vivo studies concluded that surface modiﬁcation with HA re-

inforces the implant-tissue interface, consequently improving

osseointegration.[ ] It was hypothesized in this study that the

outermost layer of HA is dissolved with time, exposing the sub-

sequent chitosan layer and improving the integration of the ti-

tanium alloy implant. HA has also found great potential to im-

prove lubrication of biomaterials at articulating interfaces (e.g.,

implants for meniscus replacement),[ ] which is vital to reduce

irritation, pain, and inﬂammation of the implant. Dopamine-

modiﬁed HA and polylysine multilayer ﬁlms were assembled on

than sulfated ones toward cancerous cells. c) Confocal images of various carboxylated nanoparticles incubated with ovarian cancer cells, demonstrating

that HA-modiﬁed nanoparticles are internalized by cells and not bound to their membrane. Cell membranes are stained in red, nuclei in blue, and

nanoparticles appear in green. Scale bar: 10 μm. d) Fluorescence images of tumor sections 24 h after nanoparticle administration in mice bearing

orthotopic ovarian cancer xenografts. Red and blue signals correspond to H&E staining, while nanoparticles appear in green (panel above). In the lower

panel, z-stacks from the whole-tumor are represented. Blue signal refers to collagen, while red signal is associated to the mCherry signal from tumor cells.

Nanoparticles appear in green. Scale bar: 100 μm. The quantiﬁcation on the z-stacks demonstrate that hyaluronic acid-coated nanoparticles penetrate

Figure 11. a) Schematic representation of the layer-by-layer approach to coat a planar substrate with hyaluronic acid (HA)/montmorillonite (MMT) and

loaded with gentamicin (GS). Enzymatic degradation in presence of hylaruonidase triggers the release of the encapsulated drug and the peeling of the

external ﬁlm. b) Gentamicin release from the multilayer ﬁlms in the presence of increasing concentrations of hyaluronidase (HAS) demonstrating the

responsiveness of the system. c) Bacterial colonies before (a,b) and after (a′,b′) incubation with the multilayer ﬁlm for 1 h. The bar graph represents the

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polycarbonate urethane substrates. The coating promoted the re-

cruitment of a lubricious glycoprotein (i.e., PRG) from synovial

ﬂuid and helped to dissipate only one-third of the frictional en-

ergy and reduce the coecient of friction with respect to the pris-

tine material.

Interestingly, metallic implants such as magnesium alloys,

have also beneﬁted from HA-based coatings.[, ] In a partic-

ular example where magnesium alloys were explored as cardio-

vascular stents, polydopamine was used as an adlayer for the

subsequent immobilization of HA via covalent interactions. The

coating prolonged the degradation time of the biomaterial, while

promoting endothelialization (i.e., increased expression of CD

of vascular endothelial cells was observed in vitro). Besides, the

blood compatibility was improved (i.e., less aggregation of red

blood cells was observed in vitro) and the inﬂammation was at-

tenuated (i.e., lower macrophage attachment in vivo) in the HA-

coated samples with respect to the pristine magnesium allow,

which opens the possibility of this material as a coronary stent.

In many occasions, biomaterials employed as cell substrates

lack in bioactivity which enormously limit their potential use as

scaolds for tissue engineering applications. Coating the surface

of these materials with extracellular matrix components such as

HA has been also considered in this ﬁeld as a strategy to im-

prove the performance of scaolds for the regeneration of sev-

eral tissues including tendon,[ ] ligaments,[ ] vocal folds,[ ]

and cartilage.[ ] For the regeneration of tendon, aligned poly(𝜖-

caprolactone) (PCL) electrospun mats were coated with HA and

chitosan via the LbL approach. In comparison to the noncoated

PCL scaold, tendon stem/progenitor cells displayed increased

expression of tenogenic markers in vitro and also in an in vivo

model of degenerative rotator cu tendon.[ ] This was ascribed

to a higher hydrophilicity and wettability provided by the multi-

layer coating, which can better resemble the conditions found in

a real cellular microenvironment.

In a dierent example,[ ] PCL electrospun microﬁbers were

functionalized with HA via LbL deposition based on bioorthogo-

nal reaction between s-tetrazines and trans-cyclooctenes, which

were coupled to the HA backbone. Moreover, the bioorthogonal

chemistry proposed in this study was fast, avoids long incuba-

tion times to create multilayer ﬁlms. The created scaolds had

interesting features for the regeneration of vocal folds since they

were able to preserve the phenotype of vocal fold ﬁbroblasts and

avoided their dierentiation toward myoﬁbroblast. This is of vital

importance to suppress ﬁbrogenesis and promoting tissue repair.

At a more basic level, the properties of HA have been exploited

to create cell substrates with controlled stiness[ ] or micropat-

terned surfaces,[ ] which enable to study important aspects of

cell biology. In the former case,[ ] poly--lysine/hyaluronic acid

multilayer ﬁlms were grown on glass slides and their stiness

was controlled by dierent crosslinking degrees via the carbodi-

imide chemistry. The stiness of the substrate clearly inﬂuenced

cell fate, being stier ﬁlms more appropriate for cell adhesion

and spreading. In the latter case,[ ] the cell-repulsive properties

of HA were used to create micropatterned surfaces that allow the

coculture of dierent cell lines with spatial resolution. HA has

also been involved in “substrate-mediated gene delivery.”[] In

a particular example, HA-based multilayer ﬁlms were used as a

platform for the encapsulation and immobilization of gene vec-

tors, allowing more localized and ecient gene delivery. Finally,

within the ﬁeld of HA as a biocoating polymer, the direct coating

of injured/damaged tissues with HA should be highlighted.[ ]

For example, a multilayer ﬁlm at the nanoscale made out of chi-

tosan and HA was directly deposited on a damaged artery. The

coating prevented blood coagulation and promoted the healing

process by inhibiting restenosis and vascular cell proliferation.

9. HA-Based Inorganic Conjugate Composites

Nanomaterials including carbon, gold, silver, magnetic and

mesoporous materials have distinct physiochemical and biologi-

cal characteristics, making them attractive for use in various drug

delivery, biosensing, diagnostic, imaging, and therapy.[] These

nanomaterials, however, are potentially cytotoxic and lack cell-

speciﬁc activities, as a result, these nanocarriers have been func-

tionalized with a variety of biopolymers including HA.[ ] Var i-

ous studies have been conducted in recent years on the develop-

ment of inorganic nanocomposites functionalization with biolog-

ical molecules, polysaccharides, biopolymers, and carbohydrates

to make them less-cytotoxic, with enhanced targetability, and cell-

speciﬁcity etc.[ ] The ability of HA to target speciﬁc cells via in-

teracting with cell surface receptors such as CD, RHAMM has

been documented by a large number of studies, and it has the

potential to be used for tumor-targeted medication delivery.[ ]

In the recent years, HA-based functionalization of inorganic

nanocomposites has gained a lot of attention for their potential

to deliver target-speciﬁc payload via cell receptor interactions with

enhanced cell-speciﬁc delivery applications. Of late, from the ma-

rine mussel inspired properties (adhesive and cohesive) of cate-

chol (CA), Lee et al. developed anticancer drug DOX loaded sil-

ica core and catechol-modiﬁed HA (HA-CA) shell delivery carrier

system with pH-triggered release behavior with enhanced cyto-

toxic activity in cancer cells, in vitro[ ] (Figure 12).

In a similar line of research HA has been explored to surface

functionalize on hybrid silica nanocarriers with enriched thera-

nostic applications. The novel organic/inorganic hybrid vesicle

(HA–docetaxel/perﬂuoro-n-pentane@silica nanocapsule–HA–

DTX/PFP@SNC) system has shown a stronger signal compared

to DTX/PFP@SNC due to high stability, ultrasound-triggered

drug release with HA-mediated tumor targeting.[ ] An attempt

has been made to increase the therapeutic ecacy of anti-

cancer drugs (including -Fluorouracil) to target colon cancer

by HA-conjugated -FU silica nanoparticles with enhanced

drug transport into tumors than normal tissues;[ ] together

these ﬁndings suggest that HA-integration on silica carriers can

be helpful in designing advanced, cell-compatible, safer drug

delivery, and contrasting agent systems. Multimodal imaging

systems based on several modalities have gained a lot of attention

in recent years for non-invasive targeted imaging applications.

Studies were reported on developing new enzyme-speciﬁc

superparamagnetic iron oxide nanoparticles applied as a multi-

modal probe in magnetic resonance imaging and hyaluronidase

(HAdase)-sensitive optical imaging applications.[ ]

Gold-hybrid nanomaterials have exhibited great promise as

nanomedicine agents due to their optical properties, includ-

ing the surface plasmon resonance eect. Recent studies have

demonstrated the formulation of gold-nanoclustered HA (GNc-

HyNA) assemblies for photodynamic tumor ablation applica-

tion, in which tumors were completely ablated with %

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Figure 12. a) Pictorial illustration of silica core with modiﬁed-HA–catechol shell formation with diﬀerent pH conditions. b) Enhanced cell-death was

Figure 13. a) Schematic representation of the HA–FA–DOX gold nanorod conjugate system for synergistic chemo and photothermal therapy. b) Com-

pared with non-FA-decorated nanoparticles, the FA–HA conjugated nanoparticles showed enhanced antitumor eﬀects against MCF-7 breast tumors in

survival rate in an orthotopic breast cancer model.[ ] In a

similar approach of multimodal targetability with photother-

mal chemotherapy application, Xu et al. developed a pH/near-

infrared (NIR) dual-triggered drug release nanocarrier system

with HA-functionalized gold nanorods (GNRs) for actively tar-

geted synergetic photothermal chemotherapy (using DOX) for

breast cancer therapy. The DOX, folate-conjugated novel GNRs–

HA–FA–DOX system showed an excellent stability with sus-

tained, dual-responsive drug release behavior with targeted syn-

ergistic breast cancer treatment[ ] (Figure 13).

In recent years, graphene oxide (GO) has attracted tremen-

dous attention for its application in anticancer therapies us-

ing HA nanohybrid conjugates. Song et al reported HA–GO–

DOX nanohybrid system, which was synthesized by conjugat-

ing GO with HA (by H bond interactions) for targeted deliv-

ery of DOX; compared to free DOX the HA–GO–DOX showed

a higher tumor inhibition eect in an H hepatic cancer cell

bearing mouse model.[ ] Similarly, HA-conjugated titanium–

graphene oxide (HA-Ti@GO) with indocyanine green (ICG)

system,[ ] MGO@CD–CA–HA hybrid system[ ] (coating of

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Tabl e 5 . Recent advancements, ﬁndings of HA–inorganic composite systems and biomedical applications.

Type of carrier Components of the delivery

system

Conjugation type In vitro/in vivo

study

Therapeutic application and ﬁndings Refs.

Silica Doxorubicin (DOX) loaded

silica core with

catechol-modiﬁed HA

(HA-CA) shell system

Amide linkage

(HA–Dopamine)

In vitro •pH-triggered release behavior with

enhanced cytotoxic activity in cancer cells

[147]

Silica HA–docetaxel/perﬂuoro-n-

pentane@silica nanocapsule

(HA-DTX/PFP@SNC)

system

Amide linkage (HA with

surface -NH2 of SNC

In vitro +In vivo •Theranostic application (imaging with

delivery)

•High stability, ultrasound-triggered drug

release with HA-mediated tumor targeting

[148]

Iron oxide Paclitaxel (PTX) loaded iron

oxide nanoparticles with

modiﬁed dopamine-bovine

serum albumin (BSA)/HA

Amide linkage

(Fe3O4·DA-BSA/HA-

PTX)

In vitro •Fe3O4·DA-BSA/HA was capable of

entrapping PTX with enhanced

bioavailability

•Time-domain (TD)-NMR experiments

revealed the system suitability as contrast

agents in MRI

[156]

Iron oxide +

graphene oxide

Doxorubicin (Dox) and

paclitaxel (Ptx) loaded

HA–GO composite with

iron-oxide nanoparticles

Amide linkage

(GO–HA)

In vitro •GO–HA–Dox/Ptx system eﬀectively killed

CD44-expressing MDA-MB-231 cells but

not BT-474 cells (no CD44 expression)

with additional magnetothermal

functionality

[157]

Fe3O4–graphene

oxide

MGO@CD-CA-HA hybrid

system (coating of

𝛽-cyclodextrin–cholic

acid–HA polymer

(CD-CA-HA) onto the

Fe3O4–graphene oxide

(MGO))

Amide linkages (HA to

CPT, HA to CD)

In vitro +In vivo •Multifunctional hybrid

photo-chemotherapy nanosystem

•Synergistic multiple targeting ability with

chemo-photothermal therapy for

hepatoma treatment

[155]

Graphene oxide HA–GO–DOX nanohybrid

system

H-bonding interactions In vitro +In vivo •Compared to free DOX the HA-GO-DOX

showed higher tumor inhibition eﬀect in

an H22 hepatic cancer cell bearing mice

[153]

Gold (Gold nanorods,

GNRs)–HA–FA–DOX system

Au–catechol bonds,

hydrazone

linkages (HA–DOX)

In vitro +In vivo •DOX, folate-conjugated novel

GNRs–HA–FA–DOX system showed

excellent stability with sustained,

dual-responsive drug release behavior

with targeted synergistic breast cancer

treatment

[152]

Titanium–

graphene oxide

HA-conjugated

titanium–graphene oxide

(HA-Ti@GO) with

indocyanine green (ICG)

drug

Amide bond In vitro +In vivo •HA-Ti@GO/ICG – multifunctional

tumor-targeted and NIR light-induced ICG

delivery system

•Signiﬁcantly increased ICG accumulation

in tumor tissue and prolonged in vivo

circulation of ICG

[154]

𝛽-cyclodextrin–cholic acid–HA polymer (CD–CA–HA) onto the

FeO–graphene oxide (MGO)) were reported for synergistic

chemo-photothermal combination therapies. Various recent ad-

vancements and ﬁndings of HA-conjugated inorganic based de-

livery systems are reported in Table 5.

10. Conclusion and Future Prospects

Although the continuation of native HA and modiﬁed-HA sys-

tems and their use in the biomedical applications has been doc-

umented for decades, it is only recently that research eorts on

HA are aimed at pharmacological, pathological, biological matrix

remodeling aspects in multiple human disease processes with

underlying mechanisms. This review has explored a variety of

chemical and biological aspects to be considered in developing

HA-based therapeutic conjugate systems for various biomedical

applications with latest research ﬁndings.

Despite positive results from several in vitro and in vivo ex-

perimental studies, it is surprising that very few clinical trials

with HA-based conjugates are being investigated. The number

of clinical trials success rate using conjugate systems in preci-

sion medicine has been quite limited in recent years. One of

the ongoing trials, ONCOFID-P-B (paclitaxel–HA conjugate) is

being studied in phase-III clinical trial to determine its eec-

tiveness and safety in patients with Bacillus Calmette-Guérin-

unresponsive bladder cancer.[,] Similarly, in another recently

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completed trial HYMOVIS (partial hexadecylamide of HA conju-

gate, clear hydrogel) device was used to evaluate the beneﬁcial

eects for knee osteoarthritis.[, ]

Besides, with the exception of Declage (developed by LG Life

Sciences in Korea) there is no commercially available drug deliv-

ery product based on HA, however, from the ongoing bioconjuga-

tion research eorts, it is expected that HA oers a great poten-

tial as a novel carrier with tunable biomimetic scaold proper-

ties for various therapeutics interventions. Nevertheless, minor

dierences in HA composition, molecular weight, type of drug

and application, and degree of conjugation alter the outcomes of

HA conjugates and must be considered when progressing those

technologies toward clinical translation.

Modiﬁed-HA systems and their biology continue to generate

interest, future studies on HA-based conjugates should focus on

including essential elements such as the utilization of HA molec-

ular weight, optimal degree of substitution, and conjugate con-

struct pharmacological and biopharmaceutical properties includ-

ing PK-PD assessments. Besides, in order to be a successful con-

jugate based product, the synthesized system should be very sim-

ple, i.e., easy preparation or scale-up, should preferably be made

of generally recognized as safe components without any toxic im-

munogenic properties. Given its unique physicochemical, phar-

macological, tissue remodeling characteristics with continuous

worldwide research eorts, HA will be successfully utilized for

the commercialization of various biopharmaceutical, medical de-

vice systems within the foreseeable future.

Acknowledgements

P.K.V. thanks Department of Biotechnology, Institute for Stem Cell Science

and Regenerative Medicine for core funds. A.L. is thankful for funds from

the Basque Government, Department of Education (IT-1766-22). I.L.M.I.

would like to thank funding agencies; Universiti Kebangsaan Malaysia un-

der the Geran Galakan Penyelidik Muda (Grant No. GGPM-2022-029),

Malaysian Ministry of Higher Education for the Fundamental Research

Grant Scheme (Grant No. FRGS/1/2022/SKK0/UKM/02/12), and Health

Research Board, Ireland under the Emerging Investigator Award for Health

2022 (Grant No. EIA-2022-010). Some of the ﬁgures were created by us-

ing BioRender.com. All authors contributed to the manuscript preparation

and approved.

Conﬂict of Interest

The authors declare no conﬂict of interest.

Author Contributions

N.G.K.: article conception; N.G.K., I.L.M.I., A.L., B.M., S.S., and G.S.: liter-

ature survey and writing; N.G.K., A.L., G.S., and P.K.V.: editing and review-

ing; All the authors: ﬁnal approval.

Keywords

bioconjugation, CD44, HA-bioconjugates, hyaluronan, hyaluronic acid

Received: November 29, 2022

Revised: March 4, 2023

Published online:

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Niranjan G. Kotla was awarded Ph.D. in biomedical engineering by University of Galway Ireland (2020)

and obtained specialist M. Pharmacy (2013), B. Pharmacy (2011) degrees. He is an innovation post-

doctoral fellow at NIBR-Novartis, Basel-Switzerland. He is an expert in drug delivery (targeted, long-

acting, controlled release), medical devices, formulation development. His research focused on

advanced drug delivery technologies development(s) using polymer chemistry,biomaterials, and

nanomedicine approaches. He is also the beneﬁciary of individual fellowships (FWO, Novartis inno-

vation postdoc), Julia Polak European doctorate awardee. He has published over 30 peer-reviewed

articles, books, conference abstracts; involved in various academic and industrial research collabora-

tions.

Gandhi Sivaraman received his M.Sc. in 2009 from The American College, Madurai, and Ph.D. in 2014

from Madurai Kamaraj University.Then he continued to pursue his postdoctoral research at Insti-

tute for stem cell science and regenerative medicine (inStem), Bangalore. Currently,he is working as

faculty in Gandhigram Rural Institute -Deemed to be University,Gandhigram, India. His research in-

terest includes chemical biology,biosensors, drug delivery systems, and computational chemistry.

His h-index is 41 and he has authored over 80 articles with over 4000 citations.

Praveen K. Vemula is an associate professor at inStem, Bangalore. He has completed master’s in

chemistry from Osmania University,and obtained Ph.D. from IISc , Bangalore. His expertise is de-

veloping chemical technologies for medical applications. His work spans the ﬁelds of biomateri-

als, drug discovery,drug delivery, medical devices, and chemical biology. He has published over 85

peer-reviewed papers and over 30 issued or pending national/international patents, which have been

licensed to multiple biotech companies. His technologies have led to the launch of six startup com-

panies. Thus far,over 20 products that are developed based on his technologies are in the market

worldwide.

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The objective of this study was to develop and characterize a novel hyaluronic acid (HA) 3D scaffold integrated with gelatin microparticles for sustained-delivery applications. To achieve this goal, the delivery microparticles were synthesized and thoroughly characterized, focusing on their crosslinking mechanisms (vanillin and genipin), degradation profiles, and release kinetics. Additionally, the cytotoxicity of the system was assessed, and its impact on the cell adhesion and distribution using mouse fibroblasts was examined. The combination of both biomaterials offers a novel platform for the gradual release of various factors encapsulated within the microparticles while simultaneously providing cell protection, support, and controlled factor dispersion due to the HA 3D scaffold matrix. Hence, this system offers a platform for addressing injure repair by continuously releasing specific encapsulated factors for optimal tissue regeneration. Additionally, by leveraging the properties of HA conjugates with small drug molecules, we can enhance the solubility, targeting capabilities, and cellular absorption, as well as prolong the system stability and half-life. As a result, this integrated approach presents a versatile strategy for therapeutic interventions aimed at promoting tissue repair and regeneration.

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Glioblastoma (GBM) is an aggressive brain tumor, with a highly immunosuppressive tumor immune microenvironment (TIME). In this work, we investigated the use of the STimulator of INterferon Genes (STING) pathway as an effective means to remodel the GBM TIME through the recruitment of both innate and adaptive immune cell populations. Using hyaluronic acid (HA), we developed a novel polymer-drug conjugate of a non-nucleotide STING agonist (MSA2), called HA-MSA2 for the in situ treatment of GBM. In JAWSII cells, HA-MSA2 exerted a greater increase of STING signaling and upregulation of STING-related downstream cyto-/chemokines in immune cells than the free drug. HA-MSA2 also elicited cancer cell-intrinsic immunostimulatory gene expression and promoted immunogenic cell death of GBM cells. In the SB28 GBM model, local delivery of HA-MSA2 induced a delay in tumor growth and a significant extension of survival. The analysis of the TIME showed a profound shift in the GBM immune landscape after HA-MSA2 treatment, with higher infiltration by innate and adaptive immune cells including dendritic, natural killer (NK) and CD8 T cell populations. The therapeutic potential of this novel polymer conjugate warrants further investigation, particularly with other chemo-immunotherapeutics or cancer vaccines as a promising combinatorial therapeutic approach.

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Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

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Hyaluronan (HA) is a naturally occurring non-sulfated glycosaminoglycan (GAG), cell-surface-associated biopolymer and is the key component of tissue extracellular matrix (ECM). Along with remarkable physicochemical properties, HA also has multifaceted biological effects that include but not limited to ECM organization, immunomodulation, and various cellular processes. Environmental cues such as tissue injury, infection or cancer change downstream signaling functionalities of HA. Unlike native HA, the fragments of HA have diversified effects on inflammation, cancer, fibrosis, angiogenesis and autoimmune response. In this review, we aim to discuss HA as a therapeutic delivery system development process, source, biophysical-chemical properties, and associated biological pathways (especially via cell surface receptors) of native and fragmented HA. We also tried to address an overview of the potential role of HA (native HA vs fragments) in the modulation of inflammation, immune response and various cancer targeting delivery applications. This review will also highlight the HA based therapeutic systems, medical devices and future perspectives of various biomedical applications were discussed in detail.

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Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve re-generation over critical defect lengths. Hydrogel luminal fillers are thought to improve the re-generation outcome by providing an optimized matrix inside the bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehen-sively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hy-drogel luminal filler did induce improved regeneration outcome over empty nerve guides. But neither combination with collagen- or chitosan-based nerve guides resulted in functional recov-ery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the crit-ical defect size model.

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Jun 2021

Skin cancer is one of the most common types of cancer in the United States and worldwide. Topical products are effective for treating cancerous skin lesions when surgery is not feasible. However, current topical products induce severe irritation, light-sensitivity, burning, scaling, and inflammation. Using hyaluronic acid (HA), we engineered clinically translatable polymer-drug conjugates of doxorubicin and camptothecin termed, DOxorubicin and Camptothecin Tailored at Optimal Ratios (DOCTOR) for topical treatment of skin cancers. When compared to the clinical standard, Efudex, DOCTOR exhibited high cancer-cell killing specificity with superior safety to healthy skin cells. In vivo studies confirmed its efficacy in treating cancerous lesions without irritation or systemic absorption. When tested on patient-derived primary cells and live-skin explants, DOCTOR killed the cancer with a selectivity as high as 21-fold over healthy skin tissue from the same donor. Collectively, DOCTOR provides a safe and potent option for treating skin cancer in the clinic.

Oligoethyleneimine‐Conjugated Hyaluronic Acid Modulates Inflammatory Responses and Enhances Therapeutic Efficacy for Ulcerative Colitis

Article

Full-text available

May 2021
ADV FUNCT MATER

Modulation of innate immune responses is a potential strategy for treating inflammatory diseases. Although biogenic polyamines are known as immunomodulatory compounds possessing anti‐inflammatory functions, their biomedical applications have been limited owing to their cytotoxicity. Here, the discovery of an anti‐inflammatory and biocompatible compound is reported, which is a relatively low‐molecular‐weight polyamine, branched oligoethyleneimine (bOEI). Among bOEI molecules, bOEI‐300 strongly suppresses the secretion of pro‐inflammatory cytokines from stimulated primary macrophages by scavenging reactive oxygen species and inhibiting the nuclear translocation of the nuclear factor kappa‐B. Moreover, its polyampholyte‐like conjugate with hyaluronic acid improves the biocompatibility of polyamines and enhanced anti‐inflammatory functions. In a murine ulcerative colitis model, the conjugates enhance therapeutic efficacy by suppressing the production of pro‐inflammatory cytokines. This polyamine‐conjugated biopolymer has enormous potential to treat inflammatory diseases via the modulation of inflammatory responses. An anti‐inflammatory oligoethyleneimine‐conjugated biopolymer for the treatment of ulcerative colitis is demonstrated. This conjugate possesses enhanced biocompatibility and anti‐inflammatory properties through scavenging of radical oxygen species and inhibition of the nuclear translocation of nuclear factor kappa‐B in macrophages. In a murine model of ulcerative colitis, this conjugate enhances therapeutic efficacy through the suppression of pro‐inflammatory cytokine production.

Role of CD44 in increasing the potency of mesenchymal stem cell extracellular vesicles by hyaluronic acid in severe pneumonia

Article

Full-text available

May 2021

Background Although promising, clinical translation of human mesenchymal stem or stromal cell-derived extracellular vesicles (MSC EV) for acute lung injury is potentially limited by significant production costs. The current study was performed to determine whether pretreatment of MSC EV with high molecular weight hyaluronic acid (HMW HA) would increase the therapeutic potency of MSC EV in severe bacterial pneumonia. Methods In vitro experiments were performed to determine the binding affinity of HMW HA to MSC EV and its uptake by human monocytes, and whether HMW HA primed MSC EV would increase bacterial phagocytosis by the monocytes. In addition, the role of CD44 receptor on MSC EV in the therapeutic effects of HMW HA primed MSC EV were investigated. In Pseudomonas aeruginosa (PA) pneumonia in mice, MSC EV primed with or without HMW HA were instilled intravenously 4 h after injury. After 24 h, the bronchoalveolar lavage fluid, blood, and lungs were analyzed for levels of bacteria, inflammation, MSC EV trafficking, and lung pathology. Results MSC EV bound preferentially to HMW HA at a molecular weight of 1.0 MDa compared with HA with a molecular weight of 40 KDa or 1.5 MDa. HMW HA primed MSC EV further increased MSC EV uptake and bacterial phagocytosis by monocytes compared to treatment with MSC EV alone. In PA pneumonia in mice, instillation of HMW HA primed MSC EV further reduced inflammation and decreased the bacterial load by enhancing the trafficking of MSC EV to the injured alveolus. CD44 siRNA pretreatment of MSC EV prior to incubation with HMW HA eliminated its trafficking to the alveolus and therapeutic effects. Conclusions HMW HA primed MSC EV significantly increased the potency of MSC EV in PA pneumonia in part by enhancing the trafficking of MSC EV to the sites of inflammation via the CD44 receptor on MSC EV which was associated with increased antimicrobial activity.

Molecular Insights of Hyaluronic Acid-Hydroxychloroquine Conjugate as a Promising Drug in Targeting SARS-CoV-2 Viral Proteins

Article

Full-text available

Apr 2021
J MOL STRUCT

In-silico anti-viral activity of Hydroxychloroquine (HCQ) and its Hyaluronic Acid-derivative (HA-HCQ) towards different SARS-CoV-2 protein molecular targets were studied. Four different SARS-CoV-2 proteins molecular target i.e., three different main proteases and one helicase were chosen for In-silico anti-viral analysis. The HA-HCQ conjugates exhibited superior binding affinity and interactions with all the screened SAR-CoV-2 molecular target proteins with the exception of a few targets. The study also revealed that the HA-HCQ conjugate has multiple advantages of efficient drug delivery to its CD44 variant isoform receptors of the lower respiratory tract, highest interactive binding affinity with SARS-CoV-2 protein target. Moreover, the HA-HCQ drug conjugate possesses added advantages of good biodegradability, biocompatibility, non-toxicity and non-immunogenicity. The prominent binding ability of HA-HCQ conjugate towards Mpro (PDB ID 5R82) and Helicase (PDB ID 6ZSL) target protein as compared with HCQ alone was proven through MD simulation analysis. In conclusion, our study suggested that further in-vitro and in-vivo examination of HA-HCQ drug conjugate will be useful to establish a promising early stage antiviral drug for the novel treatment of COVID-19.

Inorganic nanomaterials with rapid clearance for biomedical applications

Article

Jun 2021

Inorganic nanomaterials that have inherently exceptional physicochemical properties (e.g., catalytic, optical, thermal, electrical, or magnetic performance) that can provide desirable functionality (e.g., drug delivery, diagnostics, imaging, or therapy) have considerable potential for application in the field of biomedicine. However, toxicity can be caused by the long-term, non-specific accumulation of these inorganic nanomaterials in healthy tissues, preventing their large-scale clinical utilization. Over the past several decades, the emergence of biodegradable and clearable inorganic nanomaterials has offered the potential to prevent such long-term toxicity. In addition, a comprehensive understanding of the design of such nanomaterials and their metabolic pathways within the body is essential for enabling the expansion of theranostic applications for various diseases and advancing clinical trials. Thus, it is of critical importance to develop biodegradable and clearable inorganic nanomaterials for biomedical applications. This review systematically summarizes the recent progress of biodegradable and clearable inorganic nanomaterials, particularly for application in cancer theranostics and other disease therapies. The future prospects and opportunities in this rapidly growing biomedical field are also discussed. We believe that this timely and comprehensive review will stimulate and guide additional in-depth studies in the area of inorganic nanomedicine, as rapid in vivo clearance and degradation is likely to be a prerequisite for the future clinical translation of inorganic nanomaterials with unique properties and functionality.

Dendronized hyaluronic acid-docetaxel conjugate as a stimuli-responsive nano-agent for breast cancer therapy

Article

May 2021
CARBOHYD POLYM

To achieve target delivery of anti-tumor drugs with great biocompatibility into tumor tissues, a stimuli-responsive dendronized hyaluronic acid (HA)-docetaxel conjugate (HA-DTX-Dendron, HADD) was designed and prepared. The incorporation of HA in HADD improved the delivery of DTX to tumor cells with rich CD44 receptors. Enhanced biocompatibility and therapeutic outcomes were achieved using glyodendrons-modified HA and tumor microenvironment-responsive linkers in HADD. The glycodendron was connected with HA via GSH-responsive disulfide bonds, and the drug DTX was linked to the carrier via a cathepsin B-responsive tetrapeptide GFLG. This design resulted in self-assembly nanostructures for facilitating uptake of HADD by tumor cells and rapid release of DTX to exert its therapeutic effect. Compared to free DTX, HADD showed much higher tumor growth inhibition in the MDA-MB-231 tumor-bearing mice model (up to 99.71%), and no toxicity was observed. Therefore, HADD could be employed as an efficacious nano-agent for treating triple negative breast cancer (TNBC).

Is hyaluronic acid the perfect excipient for the pharmaceutical need?

Article

Apr 2021

Hyaluronic acid has become an interesting and important polymer as an excipient for pharmaceutical products due to its beneficial properties, like solubility, biocompatibility and biodegradation. To improve the properties of hyaluronic acid, different possibilities for chemical modifications are presented, and the opportunities as novel systems for drug delivery are discussed. This review gives an overview over the production of hyaluronic acid, the possibilities of its chemical modification and the current state of in vitro and in vivo research. Furthermore, market approved and commercially available products are reviewed and derivatives undergoing clinical trials and applying for market approval are shown. In particular, hyaluronic acid has been studied for different administrations in rheumatology, ophthalmology, local anesthetics, cancer treatment and bioengineering of tissues. The present work concludes with perspectives for future administration of pharmaceuticals based on hyaluronic acid.

Hyaluronic Acid‐Based Bioconjugate Systems, Scaffolds, and Their Therapeutic Potential

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