Intelligent polymeric hydrogen sulfide delivery systems for therapeutic applications

Abstract

Hydrogen sulfide (H₂S) plays an important role in regulating various pathological processes such as protecting mammalian cell from harmful injuries, promoting tissue regeneration, and regulating the process of various diseases caused by physiological disorders. Studies have revealed that the physiological effects of H₂S are highly associated with its concentrations. At relatively low concentration, H₂S shows beneficial functions. However, long-time and high-dose donation of H₂S would inhibit regular biological process, resulting in cell dysfunction and apoptosis. To regulate the dosage of H₂S delivery for precision medicine, H₂S delivery systems with intelligent characteristics were developed and a variety of biocompatibility polymers have been utilized to establish intelligent polymeric H₂S delivery systems, with the abilities to specifically target the lesions, smartly respond to pathological microenvironments, as well as real-timely monitor H₂S delivery and lesion conditions by incorporating imaging-capable moieties. In this review, we focus on the design, preparation, and therapeutic applications of intelligent polymeric H₂S delivery systems in cardiovascular therapy, inflammatory therapy, tissue regenerative therapy, cancer therapy and bacteria-associated therapy. Strategies for precise H₂S therapies especially imaging-guided H₂S theranostics are highlighted. Since H₂S donors with stimuli-responsive characters are vital components for establishing intelligent H₂S delivery systems, the development of H₂S donors is also briefly introduced.

Graphical abstract

Highlights

• •H₂S is an endogenous gasotransmitter that plays important role in regulating various physiological and pathological pathways.
• •Controlled H₂S delivery is vital since the therapeutic effects of H₂S are highly associated with its concentrations.
• •Intelligent polymeric H₂S delivery systems possess specific targeting, stimuli responsive and imaging guided capabilities, representing a strategic option for next generation of therapies.

Abbreviations

3-MST

3-Mercaptopryruvate sulfurtransferase

ACS14

2-Acetyloxybenzoic acid 4-(3-thioxo-3H-1,2-dithiol-5-yl) phenyl ester

ADSC

Adipose-derived stem cell

ADT

Anetholedithiolethione

ALG-CHO

Partially oxidized alginate

AML

Anetholedithiolethione-loaded H₂S delivery magnetic nanoliposome

APTC

2-Aminopyridine-5-thiocarboxamide

BSA

Bovine serum albumin

BSA@MnS

Bovine serum albumin modified γ-phase MnS nanotheranostic

CA

Cardiac arrest

CAP-w-FC

Diallyl trisulfide loaded gelatin capsules with foaming ability

CBS

Cystathionine-β-synthase

CD44

Cluster of differentiation 44

c-di-GMP

Cyclic-diguanylate-guanosine monophosphate

C-FE

Oxime-containing phenylalanine-glutamic acid amphiphilic dipeptides

CLSM

Confocal laser scanning microscopy

CO

Carbon monoxide

COS

Carbonyl sulfide

CP-PEG

Polyethylene glycol-grafted conjugated polymer

CPR

Cardiopulmonary resuscitation

CSE

Cystathionine-γ-lyase

CY

Cyanine

DAI

Disease activity index

DATS

Diallyl trisulfide

DATS@MION-PEG-LF

Diallyl trisulfide loaded polyethylene glycol and lactoferrin modified mesoporous iron oxide nanoparticle

DTPA

Diethylene triamine pentaacetic acid

DTT

Dithiolthiones

E. coli

Escherichia coli

EPR

Enhanced permeability and retention

ESIPT

Excited-state intramolecular proton transfer

GSH

Glutathione

GYY4137

Morpholin-4-ium-4-methoxyphenyl(morpholino)phosphinodithioate

H₂S

Hydrogen sulfide

HA

Hyaluronic acid

hMSC

Human bone marrow stromal cell

HS⁻

Hydrosulfide ion

HUVEC

Human umbilical vein endothelial cell

IBD

Inflammatory bowel disease

IDD

Intervertebral disc degeneration

IL-10

Interleukin-10

IL-6

Interleukin-6

IVIS

In vivo imaging system

K_ATPchannel

Adenosine triphosphate-sensitive potassium channel

LBL

Layer-by-layer

L-Cys

l-cysteine

LF

Lactoferrin

LPS

Lipopolysaccharide

LV

Left ventricle

MI-R

Myocardial ischemia-reperfusion

MION

Mesoporous iron oxide nanoparticle

MRI

Magnetic resonance imaging

MS

Microsphere

NF-κB

Nuclear factor κB

NHEK

Normal human epidermal keratinocytes

NIR

Near infrared

NO

Nitric oxide

NSAID

Non-steroidal anti-inflammatory drug

NTA

N-thiocarboxyanhydride

P(Lys-stat-SATO)

Poly[lysine-stat-(S-aroylthiooxime)

PA

Photoacoustic

PAH

Pulmonary arterial hypertension

PCL

Polycaprolactone

PEG

Polyethylene glycol

PLA

Poly(lactic acid)

PLGA

Poly(lactic-co-glycolic acid)

Pry-Ps

2,2′-Dipyridyl tetrasulfide

PSD

Polysulfide H₂S donor

PTT

Photothermal therapy

RA

Rheumatoid arthritis

ROS

Reactive oxygen specie

RSS

Reactive sulfur specie

S. aureus

Staphylococcus aureus

S²⁻

Sulfide ion

SA

Sodium alginate

SATO

S-aroylthiooxime

SBC

Sodium bicarbonate

SDS

Sodium dodecyl sulfate

SF

Silk fiber

S-FE

S-aroylthiooxime-functionalized phenylalanine-glutamic acid amphiphilic dipeptides

SPRC

S-propargyl-cysteine

SPRC@PLA

S-propargyl-cysteine loaded poly(lactic acid) microsphere

TA

Tetraaniline

TME

Tumor microenvironment

TNBC

Triple-negative breast cancer

TNF-α

Tumor necrosis factor alpha

UC

Ulcerative colitis

US

Ultrasound

UV

Ultraviolet

VEGF

Vascular endothelial growth factor

α-CHCA

α-Cyano-4-hydroxycinnamic acid

1. Introduction

Hydrogen sulfide (H₂S) is well known as a toxic gas and an air pollution with the characteristic smell of rotten eggs. However, research in the past two decades have shown that H₂S could be endogenously generated and is extensive distributed in human bodies, playing an important role in regulating several physiological and pathological processes by participating cellular signaling pathways including activation of adenosine triphosphate-sensitive potassium (K_ATP) channels [1], suppression of nuclear factor κB (NF-κB) signaling [2] and regulation of cellular redox [3]. Thus, H₂S was identified as one of the endogenous gaseous signaling molecules (generally called “gasotransmitter”) like nitric oxide (NO) and carbon monoxide (CO) [1,4]. Notably, H₂S is a weak acid and there is a dynamic equilibrium among H₂S, hydrosulfide ion (HS⁻) and sulfide ion (S²⁻) under physiological conditions [5] (in which S²⁻ is unlikely to be involved in the biological regulating effects as recent study suggested that S²⁻ may be not a relevant species in water [6]). It's still unclear whether H₂S or the HS⁻ or both contribute to the observed bioactivities [7]. In this review, the term “H₂S” will refer to the equilibrium mixture of these total reactive sulfur species (RSSs).

Generally, H₂S is endogenously synthesized by a number of enzymes responsible for l-cysteine (L-Cys) metabolism, including cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), 3-mercaptopryruvate sulfurtransferase (3-MST), and could also be produced through some non-catalytic routes [8]. Owing to its high lipophilicity, H₂S could freely penetrate biological membranes without any specific receptor mediation [4,9]. H₂S is also involved in several intracellular chemical reactions, which are important basis of its biological effects [8]. For example, H₂S could chelate with metalloproteins or induce S-persulfidation of cysteine residues, which in turn modulates protein activities [10,11]. In addition, H₂S participates in many intracellular redox reactions including the reduction of disulphide bonds and acts as antioxidant to scavenge free radicals [8]. Physiological concentration of H₂S usually shows a typical “protective” effect that could protect cells and tissues from harmful oxidative injures, through a combination of antioxidant and antiapoptotic signals [12]. In cardiovascular system, H₂S dilates blood vessels [13] and is of importance in cytoprotection during the evolution of myocardial ischemia-reperfusion (MI-R) injury [14]. In the nervous system, H₂S serves as synaptic modulator and neuroprotectant [12]. H₂S also promotes endothelial cell proliferation and angiogenesis [15,16], thus enhancing tissue regeneration. In addition, H₂S shows complex effects in inflammation [17] and cancers [2], and involves in some diseases such as Alzheimer's disease [18], Parkinson's disease [19] and glycometabolic disorders [20]. It should be noted that H₂S follows dose-dependent biological effects. Physiological low concentration of H₂S has beneficial and cytoprotective effects. When the biosynthesis of H₂S was inhibited, or the administration of H₂S is too excessive, the equilibrium of physiological RSSs would be changed drastically, and the stimulatory effect of H₂S would be superseded by an inhibitory effect [21].

The widespread discovery of the physiological effects of H₂S has led to the development of H₂S therapies. Inhalation therapy is the most conspicuous method for delivering exogenous H₂S and has shown protective effect against ventilator-induced lung injury [22]. However, the delivery rate and dosage of gaseous H₂S need to be strictly controlled, and the short half-life of H₂S limits the therapeutic delivery to deep lesions [23]. Compounds that could release H₂S (also known as H₂S donors) have become useful chemical tools for studying of H₂S therapies. Inorganic sulfide salts are the simplest H₂S donors and the most frequently used alternatives to gaseous H₂S during research, but they release H₂S immediately once in aqueous solution, which is difficult to mimic endogenous H₂S. To achieve controlled H₂S delivery, a series of H₂S donors activated by varying endogenous stimuli including hydrolysis, thiol containing compounds, enzyme, and exogenous stimuli like light have been developed in recent years [24,25]. These H₂S donors have prolonged release kinetics compared with sulfide salts, and most of these molecules have shown significant therapeutic potentials in a variety of biological processes [26].

Though lots of small molecular H₂S donors have been developed, they usually unable to meet the requirements of in vivo applications, since the stability, water solubility, stimuli-responsive property, and toxicity of donors themselves or their byproducts are difficult to regulate at the small molecule level [27]. With the purpose to overcome these limits, various biocompatible polymers are utilized to establish H₂S delivery systems, mainly by physically encapsulating or chemically conjugating H₂S donors with polymeric carriers such as micelles [28,29], liposomes [30], nanoparticles [31], nanofibers [32], or hydrogels [33]. The main advantage of these polymeric H₂S delivery systems is that the H₂S releasing behaviors including releasing dosage, releasing kinetics, as well as releasing locations are effectively regulated and the biocompatibility is significantly improved without drastically changing the chemical properties of the loaded H₂S donors.

More recently, against the dose-dependent therapeutic effects of H₂S, researchers have dedicated themselves to the development of H₂S delivery systems with intelligent characteristics, which integrate specific targeting, stimuli responsive and imaging guided capabilities to minimize side effects and maximize therapeutic efficacy, facilitating the precise treatment of various diseases. Through the rational design of macromolecular architectures and the utilization of suitable H₂S donors, the abilities to specifically target and selectively accumulate in lesions, as well as smartly respond to pathological microenvironments (pH [34], thiol containing compounds [35], enzyme [36], etc.) and exogenous stimuli (light [37], ultrasound [38], etc.) to release H₂S in a controlled manner could be endowed. The strategy to induce multistage in vivo reaction is promising for achieving precise and efficient H₂S therapy. Moreover, the utilization of materials with imaging capability (e.g., conjugated polymer) make it possible for imaging guided H₂S theranostics, to real-time monitor H₂S release and lesion condition.

In this review, we overview the recent advances of intelligent polymeric H₂S delivery systems for biomedical applications (summarized on Table 1), focusing on the design principles and intelligent characters of materials, as well as the physiological and pathological functions mediated by H₂S.

Table 1.

Summary of intelligent polymeric H₂S delivery systems.

Polymeric carriers	H2S donors	Intelligent abilities	Therapeutic potentials	References
Large porous microspheres	ACS14	Lung accumulation	Relief of pulmonary arterial hypertension	[39]
Peptide hydrogels	SATO	Cys responsive	Limiting the development of intimal hyperplasia in human vein segments	[40]
Hyaluronic acid and chitosan self-assembled films	ACS14	pH responsive	Regulating vascular remodeling	[41]
Conductive hydrogel	2-Aminopyridine-5-thiocarboxamide	Thiol responsive	Myocardial infarction treatment	[42]
Polymeric micelles	ADT	N/A	Protecting cardiomyocytes from ischemic cell death	[43]
PEG and lactoferrin modified mesoporous iron oxide nanoparticles	DATS	Magnetic guided, blood-brain barrier transporting, brain-targeting, MRI	Cerebral and myocardial protection after cardiac arrest	[44]
Polymeric hydrogel	α-Thioetherketone	UV responsive	Antithrombosis	[45]
SDS and SBC loaded gelatin capsule	DATS	In situ self-spray, thiol-responsive	Inflammatory bowel disease treatment	[46]
Collagen hydrogel	JK1	pH and enzyme dual-responsive	Disc degeneration treatment	[47]
Poly(lactic acid) microspheres	SPRC	N/A	Rheumatoid arthritis alleviation	[48]
PEG-ADT conjugate	ADT	N/A	Promoting inflammation	[49]
Polymeric micelles	ADT	N/A	Promoting inflammation	[50]
Polymeric nanoparticles	Arylthioamide	Thiol responsive	Angiogenesis	[51]
Polymeric micelles	ADT	N/A	Angiogenesis	[52]
Polycaprolactone nanofibers	JK1	pH responsive	Wound healing	[32]
Hyaluronic acid hydrogel	JK1	pH responsive	Wound healing	[53]
Sodium alginate sponge	JK1	pH responsive	Wound healing	[54]
Silk fibroin porous scaffolds	GYY4137	N/A	Bone tissue engineering	[55,56]
Phase-change material-loaded wound dressing	Na2S	Thermal responsive	Diabetic wound healing	[57]
Enzyme-functionalized albumin	Thiosulfate cyanide sulphurtransferase	In-situ enzymic H2S generation	Cardiac tissue repair	[58]
PEG-cholesteryl conjugate	Trisulfide	Thiol responsive	Anticancer effects	[59]
Polymeric micelles	SATO	Cys responsive	Anticancer effects	[28]
Magnetic nanoliposomes	ADT	Magnetic guided, US and MRI dual model imaging	Anticancer effects	[60,61]
BSA modified MnS nanoparticles	Metastable-phase MnS	pH responsive, MRI imaging	Anticancer effects	[62]
FeS embedded BSA nanoclusters	FeS	pH responsive, MRI imaging	Anticancer effects	[63]
F127 nanoparticles	Polysulfide	GSH responsive, ratiometric PA imaging	Triple-negative breast cancer treatment	[35]
PEG-modified conjugated polymer nanoparticles	Polysulfide	GSH responsive, NIR Ⅱfluorescence imaging	Cancer treatment, wound healing	[64]
Hyaluronated liposomes	Phenyl substituent ADT- doxorubicin conjugate	Tumor-targeted	Cancer treatment	[30]
Zwitterionic nanoparticles	L-Cys	GSH responsive	Cancer treatment	[65]
Polymersomes	SATO	Bacteria-targeted, Cys responsive	Healing of infectious diabetic wound	[66]
Peptide hydrogels	SATO	Cys responsive	Disrupting S. aureus biofilms	[67]
Polymeric microspheres	DATS	Thiol-responsive	Limb ischemia treatment	[68]
PEG-coated upconversion nanoparticles	Geminal-dithiol	NIR responsive, bioimaging	N/A	[37]
PEG brush polymers	Trisulfide	Thiol responsive	Ameliorating cellular oxidative stress	[69]
Aggregates of mPEG and cholesteryl conjugates	Trisulfide	Thiol responsive	Mitigating ROS generation	[70]
Crescent-shaped peptide assemblies	SATO	Cys responsive, enhanced cell internalization	Reducing ROS levels in macrophages	[71]
Polymeric hydrogels	SATO	Elastase-degradable, Cys responsive	Reducing toxicity of doxorubicin	[33]
Polycaprolactone microfibers	N-(benzoylthio)benzamide	Thiol responsive	Protecting cell from oxidative damage, cells proliferation	[72]
Polymeric micelles	Arylthioamide	Thiol responsive	Spatiotemporally confined cell signaling	[29]
Polymeric nanoparticles	SATO	Cys responsive, bioimaging	N/A	[73]

2. H₂S donors categorized by different activation mechanisms

H₂S donor has become a useful tool for studying therapeutic effects of H₂S and an important basis for constructing intelligent polymeric H₂S delivery systems. Thus, we discuss the classification of H₂S donors first, mainly focusing on the stimuli-responsive mechanisms and the application scopes of different donors (Fig. 1). In addition, polymeric H₂S donors have been actively explored for controlled H₂S delivery, showing enhanced stability and bioavailability over their small molecular counterparts [[74], [75], [76]]. In this part, the methods for incorporating H₂S donors with polymeric carriers would also be discussed.

Fig. 1.

Chemical structures of H₂S donors. (a) Hydrolysis-activated H₂S donors. (b) Thiols-activated H₂S donors. (c) Enzyme-activated H₂S donors. (d) Light-activated H₂S donors. (e) COS precursors as H₂S donors.

2.1. Sulfide salts as direct sources of H₂S

Sulfide salts, such as Na₂S and NaHS, are the simplest H₂S donors and currently the most used alternatives to evaluate the therapeutic potential of exogenous H₂S. Though generally classified as H₂S donors, these sulfides salts are direct sources of H₂S [24]. Once dissolved in water, they would hydrolyze and generate an equilibrium between H₂S, HS⁻ and S²⁻ immediately, and afterward a rapid volatilization of H₂S would occur. Since high-dose administration of sulfide salts is usually required for studying the biological effects of H₂S, it would cause the initial concentration of H₂S higher than physiological level and then decrease rapidly [24]. This is contradictory to the strictly modulated production of endogenous H₂S, usually cannot sustain therapeutic effects and may induce side effects. In order to achieve controlled H₂S delivery, polymeric carriers could be employed to encapsulate these sulfide salts [57,77]. Unlike Na₂S and NaHS, some other sulfide salts (MnS, FeS, ZnS, etc.) have poor water solubility, but could hydrolyze slowly to generate H₂S and metal ions under acid pH [63]. For biomedical applications, these sulfide salts could be prepared as nanoparticles and modified with biocompatible polymers [63,78,79]. The released metal ions may also show therapeutic functions in synergistic with H₂S.

2.2. Hydrolysis-activated H₂S donors

Hydrolysis-activated H₂S donors are able to release H₂S spontaneously in aqueous solution. Lawesson's reagent and its derivatives are phosphorodithioate containing H₂S donors with slow releasing property. Among them, the most well-known is morpholin-4-ium-4-methoxyphenyl(morpholino)phosphinodithioate (GYY4137), which has been utilized in many studies about the physiological effects of H₂S [80]. Another type of phosphorodithioate containing H₂S donor is JK donors possessing a unique acidic pH accelerated H₂S releasing behavior [81]. To further regulate H₂S delivery, phosphorodithioate containing H₂S donors were generally incorporate with polymeric systems through physical encapsulation [32,47,55]. Dithiocarbamates could also be activated by acidic pH to release H₂S, which have been incorporated with polymeric carriers for therapeutic application [34]. These acid pH-responsive H₂S donors are more suitable for the pathological conditions associated with acidic microenvironments, such as the sites of tumor, inflammation, and ischemic injury.

2.3. Thiols-activated H₂S donors

Many H₂S donors could be reduced by thiol containing bioactive compounds such as cysteine (Cys) and glutathione (GSH) to release H₂S. These donors are particularly suitable for controlled delivery of H₂S to the pathologically redox microenvironments. N-(benzoylthio)benzamides developed by Xian et al. [82] undergo a thioester exchange reaction to form N-SH intermediates, which further react with these compounds to produce H₂S. S-aroylthiooximes (SATOs) developed by Matson et al. [83] could react with Cys-containing compounds to form thiooxime intermediates that further react with these compounds to generate H₂S. The H₂S releasing profile of the above two donors could be regulated by tuning the substituents of donor aromatic rings. Furthermore, thiooxime formation reaction could be conducted at mild condition with high efficiency, thus SATOs have been utilized to establish polymeric H₂S donors through postpolymerization approaches [28,73]. Naturally occurring polysulfides, represented by diallyl trisulfide (DATS), undergo a thiol-promoted cleavage of polysulfide bond and form hydrosulfide intermediates, which further react with thiol containing compounds to produce H₂S [84]. Polysulfide linkers have been introduced to the backbones or side chains of polymers to enable controlled H₂S delivery for various biological applications [59,69,70,85]. Synthetic acyl perthiols are also S-S bond containing H₂S donors [24] and have been modified on the end group of polymers [86]. In addition, arylthioamides could release H₂S in response to both thiol containing compounds and hydrolysis, although its chemical mechanism is unclear [87]. Dithiobenzoate end group of polymers synthesized through reversible addition fragmentation chain transfer polymerization may themselves be H₂S donors upon exposure to thiol containing compounds [88]. In comparison with these thiol containing compounds specific H₂S donors, dinuclear persulfide-bridged ruthenium complex could be activated by a variety of reducing agents including HSO₃⁻, Cys, GSH and ascorbate to release H₂S, showing unique advantage in hypoxia conditions [89].

2.4. Enzyme-activated H₂S donors

Utilizing enzyme-activated H₂S donors has many advantages for targeted H₂S delivery to enzyme-overexpressed microenvironments of lesions. Trimethyl lock group containing H₂S donors possess typical esterase-activated characteristics [36]. The ester-protected structure could be enzymatic hydrolyzed by esterase to produce nucleophilic hydroxy group, which further attack the carbonyl group and undergo a lactonization reaction to produce H₂S. In addition, the bis(4-nitrobenzyl)sulfanes could be reduced by nitroreductase and produce hydroxylamino- or amino-aryl derivatives to generate geminal dithiols through self-immolation, which could be further hydrolyzed to generate H₂S [90]. Dithiolthiones (DTTs), represented by anetholedithiolethione (ADT), were generally considered as hydrolysis-activated H₂S donors [91]. However, recent studies have revealed that H₂S release of ADT could occur through an enzymatic process in the presence of liver microsomes and reduced nicotinamide adenine dinucleotide phosphate [92]. ADT could be conjugated with polymers to overcome the poor solubility and possible side effects [49]. Naturally occurring glucosinolates could be hydrolyzed under the catalysis of myrosinase [93]. One of the possible products depending on glucosinolate side chains and reaction conditions, isothiocyanate, could act as H₂S donors [94]. A more recent study demonstrated that 1-thio-β-D-glucose could act as a H₂S/H₂O₂ dual donor catalyzed by glucose oxidase, which could cause efficient protein S-persulfidation synergistically, since thiol groups of proteins could be oxidate by H₂O₂ to generate sulfenic acids (RSOH) that could react with H₂S for persulfidation more efficiently [95].

2.5. Light-activated H₂S donors

In recent years, strategies using exogenous stimuli as triggers to regulate H₂S delivery are being actively developed. Among these exogenous stimuli, light could trigger the H₂S release without perturbing native biochemical process, minimize off-target effects though direct spatiotemporal control, showing great advantages in the tissue-specific delivery of H₂S [24]. Light-activated H₂S donors usually contain photoremovable protecting groups, which could be deprotected under light irradiation at appropriate wavelength to release H₂S. o-Nitrobenzyl caged geminal dithiols [96] and ketoprofenate caged H₂S donors [97] are reported to release H₂S under ultraviolet (UV) light. In addition, α-thioetherketones are UV light-activated prodrugs of thioaldehydes, which could further release H₂S in the presence of amines [45]. p-Hydroxyphenacyl photocaged H₂S donors could respond to visible light to release H₂S [98]. Meanwhile, the light-induced deprotection activates the excited-state intramolecular proton transfer (ESIPT) process, which leads to a distinct fluorescent emission color change (green to blue) for real-time monitoring. However, currently developed H₂S donors are mostly activated by UV or visible light with short wavelength, showing poor tissue penetration ability and potential injury to normal tissues. Thus, H₂S donors triggered by physiological-friendly and tissue-penetrable visible or near infrared (NIR) light need to be further developed.

2.6. Carbonyl sulfide (COS) precursors as H₂S donors

COS could be quickly hydrolyzed to H₂S by carbonic anhydrase that is ubiquitously distributed in human tissues. The connection between COS and H₂S has led to the study on COS generating compounds as H₂S donors for biological applications. Matson et al. reported that N-thiocarboxyanhydride (NTA) could generate COS upon ring-opening by a biological nucleophile [99]. In addition, the strategies utilizing S-alkyl or O-alkyl thiocarbamate or thiocarbonate based self-immolative linkers, initially proposed by Pluth et al. [100], have been employed for customizing COS donors, since COS release is the result of the self-immolative reactions, and the activation mechanism could be tuned by altering the protecting groups. Specifically, aryl boronate ester linked thiocarbamate/thiocarbonate could response to reactive oxygen species (ROSs) to activate self-immolative process to generate H₂S via COS [101,102]. When protected by imine, acidic pH activated COS release could be achieved upon imine cleavage [103]. When para-pivaloyl group or pivaloyloxymethyl group are linked, the donors could be activated by esterases [104,105]. When photoactivated groups such as o-nitrobenzyl [106] or boron dipyrromethene chromophores with different substituents [107,108] were linked, UV to NIR light activated H₂S release could be achieved. In addition, through the linkage of appropriate fluorophores, self-reporting release of COS could also be achieved for real-time monitoring [[109], [110], [111], [112]]. Furthermore, an oligo(thiourethane) containing S-alkyl thiocarbamate linkers was synthesized through the polyaddition of 4-isothiocyanatobenzyl alcohol and end-capped with aryl azide, which could undergo self-amplified depolymerization to release multiple equivalents COS in response to per equivalent of H₂S [113].

3. Therapeutic applications of intelligent polymeric H₂S delivery systems

3.1. Cardiovascular therapy

H₂S has demonstrated critical effects on cardiovascular system [114]. Study on isolated blood vessels exhibited that H₂S could increase the K_ATP channel currents of smooth muscle cells and hyperpolarize the membrane, thus relaxing smooth muscles, dilating blood vessels and reducing blood pressure [13]. Additionally, H₂S was reported to exhibit significant cardioprotective ability by attenuating MI-R injury [14]. The process of reperfusion after myocardial ischemia could cause severe oxidative damage to myocardial cells due to the generation of ROS [115]. Under the oxidative stress during MI-R, H₂S could act as a potent antioxidant, and up-regulate the GSH production to protect myocardial cells from oxidative damage together [12]. In addition, H₂S could inhibit leukocyte adherence to reduce myocardial inflammation, regulate mitochondrial respiration to preserve mitochondrial function, and inhibit the apoptosis of cardiomyocytes during MI-R injury [14].

Several research teams have started the exploration of exogenous H₂S delivery for treating cardiovascular diseases. Xian et al. developed a series of controllable H₂S donors and evaluated the cardioprotective effects against MI-R injury [81,116,117]. Compared with small molecular H₂S donors, nanosized polymeric H₂S delivery systems exhibit more excellent cardioprotective properties. Matson et al. reported that SATO-bearing peptide assemblies could rescue H9C2 cardiomyocytes from doxorubicin-induced cytotoxicity [118]. Hasegawa et al. showed that ADT-containing polymeric micelle could release H₂S intracellularly and prevent cardiomyocyte apoptosis in an in vitro ischemia model, more effective than NaHS and ADT-OH [43]. Sun et al. loaded the polysulfide H₂S donor DATS on the polyethylene glycol (PEG) and lactoferrin (LF) modified mesoporous iron oxide nanoparticles (MIONs) to form DATS@MION-PEG-LF [44]. PEG was used to acquire the prolonged circulation time, and LF could help nanoparticles to across the blood brain barrier and gain brain-targeting effects. The DATS@MION-PEG-LF showed prominent protective effects against cerebral and cardiac ischemic injury after cardiac arrest (CA), which was proved by in vitro hypoxia/reoxygenation models and in vivo CA/cardiopulmonary resuscitation (CPR) models. The treatment process could also be non-invasively traced through magnetic resonance imaging (MRI).

M-IR injury usually arises in patients suffering from acute ST-segment elevation myocardial infarction, and limiting the size of myocardial infarction is the main treatment strategy currently [115]. H₂S is a potent regularizing factor that could significantly reduce the myocardial infarcted size and improve survival rate in acute myocardial infarction [119,120]. Liu et al. reported a stem cell loaded conductive hydrogel with H₂S delivery capacity for myocardial infarction treatment (Fig. 2a) [42]. ALG-TA-APTC copolymer was formed by grafting H₂S donor 2-aminopyridine-5-thiocarboxamide (APTC) and tetraaniline (TA) oligomer onto partially oxidized alginate (ALG-CHO), for endowing H₂S delivering capability and reestablishing the electrical impulse signals between myocardial cells in the infarcted area. Hydrogel was fabricated via covalent cross-linking of gelatin, ALG-CHO and ALG-TA-APTC, exhibiting controlled H₂S release in vitro (Fig. 2d). This hydrogel showed outstanding bioadhesive property (Fig. 2b), which could ensure a stable anchoring to the wet and beating hearts and enhance the retention of adipose-derived stem cells (ADSCs) in the MI zone (Fig. 2c) to promote the regeneration of myocardium. After hydrogel injection, an elevated H₂S concentration in rat myocardium was observed, accompanied by the increase of cardiac-related mRNA and angiogenic factors and the decrease of inflammatory factor, tumor necrosis factor alpha (TNF-α). Furthermore, the reduction in cardiac fibrosis area (Fig. 2e) and the mitigatory of left ventricle (LV) wall thinning (Fig. 2f) demonstrated the therapeutic potential of this H₂S delivery hydrogel for reducing infarction size and improving cardiac functions.

Fig. 2.

Structure, preparation, and therapeutic properties of H₂S delivery conductive hydrogel. (a) Scheme illustrating the formation of ADSC-loaded H₂S delivery conductive hydrogel. (b) Photograph showing the potent adhesive property of ALG-CHO/ALG-TA-APTC/Geln hydrogel. (c) Stem cell retention in the MI zone after injection. (d) H₂S-releasing profile in vitro, in which macromolecular H₂S prodrug embedded hydrogel showed the slowest H₂S release. (e) Fibrosis area and (f) LV wall thickness after treatment of different hydrogels (* indicates a significant difference between the experimental group and the MI group, *p < 0.05, **p < 0.01) [42]. Copyright 2019, American Chemical Society.

Pulmonary arterial hypertension (PAH) is a cardiovascular disease characterized by a persistent elevation in pulmonary vascular pressure and pulmonary vascular remodeling, which may cause right ventricular failure and even death under some circumstances [121]. H₂S has been proven to reduce pulmonary vascular remodeling via promoting the apoptosis of pulmonary artery smooth muscle cells and inhibiting collagen deposition [122]. Though gaseous H₂S could be delivery to the lung through inhalation, it is difficult to mimic slow and constant H₂S release in vivo in this way. To overcome this problem, Zhang et al. developed a H₂S delivery poly(lactic-co-glycolic acid) (PLGA)-based large porous microspheres (MSs) for inhaled therapy of PAH [39]. PLGA-based porous MSs was prepared through microfluidic technology, and 2-acetyloxybenzoic acid 4-(3-thioxo-3H-1,2-dithiol-5-yl) phenyl ester (ACS14), a H₂S donor comprising of a dithiolethione conjugated with aspirin, was loaded to form ACS14 MSs. The high porosity made ACS14 MSs light enough to reach deeply into the lungs via inhalation, and the <5 μm of mass median aerodynamic diameter was suitable for deposition in alveolar region. The sustained release of ACS14 for up to seven days was observed in simulated lung fluid. Furthermore, H₂S was generated slowly and continuously in lung tissues after the inhaled administration of ACS14 MSs, reaching maximum in 24 h and showing negligible effect on the plasma level of H₂S. In vivo fluorescence imaging of rats showed that ACS14 MSs had the characteristics of long-term lung retention and passive lung targeting. In a rat model of monocrotaline-induced PAH, the therapeutic effect of ACS14 MSs through inhalation was significantly higher than the corresponding systemic delivery of free ACS14, equivalent to sildenafil that is conventionally employed in PAH treatment.

Atherosclerotic stenosis could cause various fatal cardiovascular diseases and interventional therapy is the most common treatment method for atherosclerotic stenosis, showing considerable effectiveness in clinic. However, this therapy could damage the health tissues which may cause complications including inflammation, intimal hyperplasia, late thrombosis, and in-stent restenosis. H₂S has been found to relief vascular inflammatory response, vascular remodeling, and thrombosis at atherosclerotic sites. On account of the weak acidic microenvironment at the angioplasty site with inflammation, Huang et al. prepared a pH-responsive layer-by-layer (LBL) self-assembled film to coat on the implant surface to achieve controlled delivery of H₂S (Fig. 3a) [41]. The LBL coating was fabricated on the surface of dopamine-modified stainless-steel implants, through the sequential immersion in catechol chitosan solution, catechol hyaluronic acid solution and ACS14 solution for several rounds. Under weak acidic microenvironment, the stability of LBL coating was changed, resulting in the enhanced release of ACS14 for over 60 days (Fig. 3b and c). Ex vivo experiments on isolated arteries and veins of rabbits showed that coatings loaded with 10 μmol/L of ACS14 possessed good blood compatibility, significantly inhibiting red blood cell and platelet adhesion (Fig. 3d), which was also confirmed by reduced blood flow obstruction (Fig. 3f) and thrombus weight (Fig. 3g). To evaluate the in vivo therapeutic effects of the H₂S delivery LBL coating, ACS14-loaded LBL-coated filaments were implanted into the abdominal aorta of SD rats. No significant thrombus clogging, and inflammation were observed after 30 days, providing a novel method to solve in-stent restenosis.

Fig. 3.

Preparation of H₂S delivery coating and its therapeutic effects as cardiovascular implant. (a) Intelligent coating release H₂S in a weakly acidic for regulating vascular remodeling. H₂S donor release profiles in (b) pH 6.5 and (c) pH 7.4. (d) Coagulation of different samples with varying content of H₂S donor after ex vivo experiments on isolated arteries and veins of rabbits. (e) Schematic diagram of the ex vivo experiments. (f) Blood flow obstruction rate and (g) thrombus weight after ex vivo experiment (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001) [41]. Copyright 2021, Elsevier Ltd.

3.2. Inflammatory therapy

Inflammation is an adaptive response of innate immune system triggered by noxious stimuli to eliminate tissue injury and initiate tissue repair [123]. Conventionally, inflammation is beneficial for restoring homeostasis, lasting for only a short period of time before the body returns to health. However, when this physiological process persisted abnormally, it could lead to inflammatory diseases such as rheumatoid arthritis (RA). There is increasing evidence that H₂S acts as an endogenous modulator for resolution of inflammatory response [124]. Unlike CO that shows definite anti-inflammatory effects [125,126], the role of H₂S in inflammation is complex. Studies have shown that H₂S exerts anti-inflammatory effects by reducing the expression of many pro-inflammatory cytokines, chemokines and enzymes to inhibit the activation of NF-κB pathways, suppressing the leukocyte adhesion and recruitment, driving macrophage differentiation towards the anti-inflammatory M2 phenotype, and inducing apoptosis in neutrophils [124]. H₂S could also reduce the formation of edema [127] and relieve pain caused by inflammation [128]. It has been reported that H₂S donor-conjugated non-steroidal antiinflammatory drugs (NSAIDs) exhibit improved efficacy and reduced toxicity compared with the parent NSAIDs [129]. However, H₂S could also exhibit pro-inflammation effects on a number of pathological processes [130]. The reduced inflammatory response could be observed after treatment of H₂S synthase inhibitors [131]. Hasegawa et al. reported that PEG-ADT conjugate and ADT-containing micelle capable of delivering H₂S could enhance the pro-inflammatory cytokine TNF-α [49,50]. The contradictory effects of H₂S in inflammation observed during studies probably associate with its dose-dependent activity and varying roles at different stages of inflammation, which still needs to be further elucidated. The anti-inflammatory effect of intelligent polymeric H₂S delivery systems is the main focus of current studies, as well as the major content of this section.

RA is a complex autoimmune and inflammatory disease that could cause severe damage to joint tissues, with subsequent high morbidity and mortality. In order to alleviate RA, Zhu et al. developed a poly(lactic acid) (PLA)-based microsphere loaded with S-propargyl-cysteine (SPRC), an endogenous H₂S modulator that could stimulate H₂S generation in vivo via CSE/H₂S signaling pathways, named SPRC@PLA [48]. After subcutaneous injection of SPRC@PLA, a sustained elevation of plasma H₂S concentration was observed, different with the rapid production and fast decrease of H₂S by injecting free SPRC. Further research showed that SPRC@PLA could increase the expression of CSE to enhance the generation of H₂S, and alleviate paw swollen in adjuvant-induced arthritis rat. Intervertebral disc degeneration (IDD) is also highly associated with inflammation. Against the characteristic pathological environment of IDD including overexpressed proteolytic enzymes and acidic pH due to the accumulation of lactic acid as a result of glycolysis, Xiao et al. developed an enzyme and pH dual-responsive H₂S delivery hydrogel for IDD treatment [47]. JK1, a H₂S donor that could undergo an acidic pH-accelerated hydrolysis to release H₂S, was encapsulated in collagen hydrogel to form Col-JK1. Collagen could be gradually degraded by the highly expressed matrix metalloproteinases in IDD to release JK1, and subsequently the acidic pH within the disc could trigger the generation of H₂S in situ. Col-JK1 exhibited better therapeutic efficacy to annular puncture-induced IDD in rats than free JK1 as observed through MRI. Further study revealed that Col-JK1 could inhibit the apoptosis of nucleus pulposus cells and attenuate the degradation of the disc extracellular matrix to protect the disc from degeneration. The protective effect of Col-JK1 was attributed to its anti-inflammatory effects through the deactivation of the NF-κB signaling pathway, which could be demonstrated by the decreased expression of inflammatory cytokines interleukin-6 (IL-6) and TNF-α, as well as the increased expression of anti-inflammatory cytokines interleukin-10 (IL-10).

Inflammatory bowel diseases (IBDs), represented by ulcerative colitis (UC) and Crohn's disease, are chronic relapsing disorders of the gastrointestinal tract characterized by overexpression of pro-inflammatory cytokines, as well as adhesion of leucocytes to the vascular endothelium and their migration to the inflamed bowel. The garlic-derived natural H₂S donor, DATS, exhibited anti-inflammatory effects in both in vitro and in vivo studies [132,133]. However, due to its poor water solubility, appropriate administrating strategies are still required for improved therapeutic effects. In order to treat IBD using DATS, Sung et al. [46] developed a in situ self-spray coating system based on DATS loaded gelatin capsules with foaming ability (CAP-w-FC), which were composed of a mixture of acid initiator (diethylene triamine pentaacetic acid, DTPA), foaming agent (sodium bicarbonate, SBC), surfactant (sodium dodecyl sulfate, SDS) and DATS (Fig. 4a). CAP-w-FC was rectally administered to rat with UC. After the gelatin capsule was dissolved in the intestinal fluid, the acid initiator reacted with water rapidly to yield an acidic environment, inducing decomposition of foaming agent SBC to produce CO₂ bubbles. The bubbles were stabilized immediately by a self-assembled nanofilm of SDS, which subsequently served as a carrier for hydrophobic DATS. After the bubble carrier rose to the water/air interface and burst, the atomized DATS-loaded SDS micellar particles were formed and sprayed on the luminal surface of the colorectal tract. This process was proved by fluorescence labeling experiment in simulated intestinal fluid (Fig. 4b). In the simulated in vivo release experiment, the fluorescence signal of H₂S fluorescence probe showed that DATS was uniformly internalized by colonic epithelial cells and converted to H₂S, as observed through confocal laser scanning microscopy (CLSM) fluorescence images (Fig. 4c). Meanwhile, the pro-inflammatory cytokines, including TNF-a, MCP-1, and IL-6, were decreased and macrophage adhesion was inhibited in lipopolysaccharide (LPS)-induced RAW264.7 cells (Fig. 4e). In vivo experiments were carried out in a rat model of UC, showing that CAP-w-FC reduced the level of ROS in the colon and alleviated the inflammatory response, as observed by in vivo imaging system (IVIS) (Fig. 4d). Significantly reduced disease activity index (DAI) score was also observed after treating by CAP-w-FC (Fig. 4f).

Fig. 4.

Formulation, mechanism of action of in situ self-spray coating system, as well as its anti-inflammation effects in vitro and in vivo. (a) Schematic illustrations of dispersion of a coating of DATS-loaded micellar particles on luminal surface of colon to repair colonic inflamed tissues. (b) Fluorescence images and schematic illustrations of formation of bubble carriers and their transformation to DATS-loaded micellar particles that are stabilized by SDS. (c) CLSM fluorescence images of H₂S production in Caco-2 cells. (d) IVIS images of L-012-derived luminescence signals, showing inflamed sites in colon and their corresponding intensities in IBD rats. (e) Expression levels of TNF-a, MCP-1, and IL-6 in LPS-induced RAW264.7 cells following various treatments (*p < 0.05). (f) DAI scores of IBD rats following various treatments [46]. Copyright 2018, Elsevier Ltd.

3.3. Tissue regenerative therapy

H₂S has been reported to promote the proliferation and migration of endothelial cells, as well as activate the vascular endothelial growth factor (VEGF) receptors and the K_ATP channel, to facilitate angiogenesis, which is vital to the tissue regenerative process [16]. Meanwhile, the inflammation modulating and oxidative stress suppressing abilities of H₂S also contribute to its promotional role in tissue regeneration [53,72]. In recent years, intelligent polymeric H₂S delivery systems have started to show their unique benefits in tissue regenerative therapy. Hasegawa et al. studied the pro-angiogenic effects of ADT-containing H₂S delivery micelles which were prepared from amphiphilic block copolymers consisting of a hydrophilic poly(N-acryloyl morpholine) block and a hydrophobic block containing ADT groups [52]. The micelles could be internalized by human umbilical vein endothelial cells (HUVECs) and release H₂S intracellularly, as observed by CLSM. In the gap closure migration assay on HUVECs, ADT-containing micelles showed significant effects on cell migration, similar with the group treated by the growth factor VEGF₁₂₁ and higher than ADT-treated group. The micelles also showed significant promoting effects on endothelial cell tube formation and vascularization in the in ovo chick chorioallantoic membrane assay. Another gasotransmitter, NO, could also induce angiogenesis, showing a synergistic effect with H₂S in cell signaling pathways [134]. Thus, the angiogenesis effect is expected to be amplified by establishing H₂S and NO co-delivery systems. Lee et al. reported a H₂S and NO co-delivery nanoparticle formed by the self-assembly of thiobenzamide-functionalized methoxy poly(ethylene glycol-b-lactic-co-glycolic-co-hydroxymethyl propionic acid), loading N-diazeniumdiolated diethylenetriamine as NO donor [51]. This co-delivery system showed significantly enhanced in vitro tube formation effect on HUVECs and ex vivo angiogenesis effect on rat aorta, compared with the only NO or H₂S delivery groups.

Minor injuries in healthy individuals could generally heal well [135]. However, when it comes to open wounds caused by wars or accidents and chronic wounds such as diabetic foot ulcers, the self-healing processes may not work very well [136]. Polymeric wound dressings based on hydrogels, fibrous membranes, sponges, etc., are able to create a moist environment around the wound, facilitating the regeneration of skin tissues. Wang et al. developed a series of polymeric H₂S delivery wound dressing and managed to elucidate the mechanisms in wound healing acceleration [32,53,54,72]. According to the acidic pH at the acute wound tissues, they fabricated a JK1-loaded pH-controllable H₂S delivery polycaprolactone (PCL) fibrous material as wound dressing [32]. When lowered the pH from 7.4 to 6.0, the faster H₂S release from PCL-JK1 was observed. In the full-thickness cutaneous wound model on mice, the healing rate of the PCL-JK1 treated group was significantly higher than PCL treated group at all time points studied. Histological analysis on day 20 showed that PCL-JK1 treated group have newly regenerated tissues with fully developed granulation and re-epithelialization, as well as more and mature newly formed vascularization toward the wound compared with PCL treated group. In their following study, JK1 was doped in hyaluronic acid (HA) hydrogel to form a wound dressing (HA-JK1) [53]. Since HA is a major constituent of extracellular matrix, this hydrogel highly showed biocompatibility for in vivo wound healing. HA-JK1 significantly accelerated the wound regeneration process through enhanced re-epithelialization, collagen deposition, angiogenesis, and cell proliferation. One important observation is that this H₂S delivery hydrogel was able to induce the in situ polarization of macrophages from inflammatory M1 phenotype to pro-healing, anti-inflammatory M2 phenotype, thus reducing inflammation around the wound and improved wound remodeling effects. More recently, towards the wounds with heavy exudate levels, the same research group developed a wound dressing by incorporating JK1 into a sodium alginate (SA) sponge (Fig. 5a) [54]. The SA-based sponge could not only absorb wound exudate effectively to form hydrogel and maintain a moist environment but could also release H₂S continuously to the wound bed in response to acidic pH. The SA-based sponge could reach the maximum water uptake in approximately 1 h with high swelling ratios to promote the drainage of exudates. Additionally, the dimeters of the SA-based sponge were barely changed after swelling, showing excellent dimension stability to avoid wound laceration. H₂S release under acidic pH was accelerated as measured through methylene blue assay (Fig. 5b). Accordingly, in vitro cell scratch assay exhibited improved fibroblast proliferation and migration at pH 6.0 than at pH 7.0. Moreover, the in vivo assay using a full thickness dermal defect model revealed that the sponge could significantly improve wound healing process (Fig. 5c–e).

Fig. 5.

Acidic pH accelerated H₂S release from the SA sponge and its promoting effect on wound healing. (a) Diagram illustrating the pH dependent H₂S delivery SA sponge dressing. (b) H₂S release profiles of H₂S donor loaded SA (SA/JK-1) and donor alone (JK-1) in pH 6.0 and pH 7.4, in which acidic pH accelerated H₂S generation was observed. (c) Wound healing property of sponge dressing in mice model, in which SA/JK represented the control sponge loaded with a JK compound that cannot release H₂S. (d) Closure rate of wounds, ****p < 0.0001, n = 4–6. (e) Images showed the representative collagen deposition of wounds [54]. Copyright 2020, Elsevier Ltd.

3.4. Cancer therapy

Similar to the physiological roles in inflammatory response, H₂S exhibits complex effects on cancer [2]. H₂S could act as a bioenergetic stimulator to promote glucose uptake and glycolysis efficiency to provide the energy of cancer cells. Furthermore, H₂S is able to promote the angiogenesis, activate the anti-apoptotic pathways and accelerate the cell cycle of cancer cells to facilitate cancer development. The excessive generation of H₂S has been demonstrated in numerous types of cancer cells, which has also become potential targets in anticancer therapy to develop H₂S-triggered theranostic nanoagents for the diagnosis and treatment of cancer [[137], [138], [139], [140], [141], [142]]. However, this strategy is beyond the scope of our review.

When high-dose and long-term administration of exogenous H₂S were performed, anticancer effects are usually observed. The anticancer property of H₂S could be partly attribute to the uncontrolled cellular acidification caused by the H₂S-mediated glycolysis enhancement [2]. In addition, H₂S could suppress the cell signaling pathways that are abnormally activated during cancer development, such as NF-κB pathways [143]. H₂S could also induce cell cycle arrest in several cancer cell lines and lead to cell apoptosis [144]. Several in vitro studies exhibited that exogenous H₂S seems to work specifically on cancer cells, with no obvious impacts on normal cells. In 2017, Matson et al. reported a H₂S delivery polymer micelle formed by SATO-containing amphiphilic block copolymers, which could concentration-dependently reduce the survival of HCT116 colon cancer cells, but led to no significant effect on the viability of NIH/3T3 fibroblasts [28].

Notably, conditions of the tumor lesions in the patient's body differ greatly from that of cancer cells in vitro. Therefore, in order to enhance in vivo anticancer effects, intelligent polymeric H₂S delivery systems should be formulated based on the pathological conditions of tumor lesions. On the one hand, the enhanced permeability and retention (EPR) effect of the abnormal tumor neovasculature and highly expressed targets in cancer cells allow the targeted delivery of polymeric nanocarriers in passive and active manners [145]. Cluster of differentiation 44 (CD44), a cell surface protein overexpressing in cancer cells to promote tumor growth and metastatic dissemination, could act as a target of HA [146]. HA-modified liposome encapsulating H₂S donor-doxorubicin conjugate developed by Riganti et al. could effectively induce cell death against doxorubicin-resistant osteosarcoma cells, which was attributed to the alteration of drug delivery to endoplasmic reticulum, sulfhydration and ubiquitination of protein, and activation of endoplasmic reticulum stress pro-apoptotic response [30]. On the other hand, stimuli in characteristic tumor microenvironment (TME) including low pH, altered redox potential, hypoxia, hyperthermia, etc. [147] could be utilized to regulate H₂S delivery by modulating macromolecular architectures. A TME responsive zwitterionic H₂S delivery system was recently reported by Wan et al., which was formed through the polymerization of zwitterionic sulfobetaine methacrylate monomer using N,N′-bis-(acryloyl) cystamine as crosslinker and the subsequent loading of L-cysteine (L-Cys) and α-cyano-4-hydroxycinnamic acid (α-CHCA) [65]. It could be degraded by high concentration of GSH in TME to release L-Cys and α-CHCA. L-Cys could act as a substrate of highly expressed CSE in TME to produce H₂S, which subsequently promoted the uptake of glucose to induce tumor cell acidosis. Meanwhile, α-CHCA destroy the lactic acid transmission chain of tumor cells and cause excessive intracellular accumulation of lactic acid, leading to the destruction of tumor metabolism symbiosis and the ultimate cell death. More excessive H₂S and α-CHCA were released after incubation with MCF-7 cancer cells than normal endothelial cells HUVECs, demonstrating the good responsiveness to TME. Compared with the steady intracellular pH value of HUVECs (about 7.1), intracellular pH value of MCF-7 cancer cell deceased significantly to 6.1 due to lactic acid accumulation as a product of glycolysis. A significant decrease in ATP production was also observed in the treated cancer cells due to the disorder of cell metabolism. These results demonstrated the selective toxicity of this TME responsive H₂S delivery system.

In addition to the targeted and TME responsive polymeric H₂S delivery systems, the utilization of external stimuli (light and magnetic field, etc.) to regulate H₂S delivery temporally and spatially to the deep regions of tumors has been proven to be of great efficiency, and advanced imaging technologies facilitate the precise and visual cancer diagnosis and treatment. Inorganic iron- or manganese-based contrast agents, have been widely studied in cancer theranostics through MRI, showing enhanced biosafety than conventional gadolinium-based contrast agents. Biocompatible polymers are usually utilized for modification of these contrast agents. For example, Huang et al. developed a bovine serum albumin (BSA) modified γ-phase MnS nanotheranostic (BSA@MnS), which could be dissociated in acidic tumor microenvironment, releasing H₂S and Mn²⁺ ions simultaneously [62]. The released Mn²⁺ could not only act as contrast agent for MRI, but also catalyze Fenton-like reaction to convert H₂O₂ into toxic hydroxyl radical at tumor tissues, showing synergistic anticancer effects with H₂S. Besides, Gu et al. developed a ADT-loaded H₂S delivery magnetic nanoliposome (AML) with MRI and ultrasound (US) dual-model imaging capability for cancer theranostics by loading ADT in the phospholipid membrane and small superparamagnetic nanoparticles in the core of the liposome (Fig. 6a) [60]. AMLs could be ingested by HepG2 cancer cells and generate H₂S bubbles intracellularly, behaving like bubble bombers to physically destroy the cells (Fig. 6b). Due to the incorporation of superparamagnetic nanoparticles, the tumor targeting of AMLs could be enhanced under external static magnetic field, and the specific intratumoral distribution of AMLs could be dynamically monitored through MRI (Fig. 6d). In addition, the in situ generated H₂S bubbles could sensitize the ultrasound (US) imaging signal, to monitor the dynamic process of intratumoral H₂S production (Fig. 6c). Furthermore, therapeutic US intensity could disrupt the H₂S bubbles and enhance the physical bombing effect on tumors. The intratumorally generated high concentration of H₂S could rapidly travel through deep tumor membrane barrier to enhance antitumor effect. In their following research, the mechanisms of tumor cell death induced by H₂S bubbling were elucidated [61]. The gradual generated H₂S bubbles opened the calcium channel of cancer cells and enhanced the intracellular calcium concentration, which could interrupt the intracellular ion microenvironment (Fig. 6e). The bubble breakage also induced the generation of hydroxyl radicals, significantly influencing the intracellular redox homeostasis in tumor cells. In addition to affecting the tumor cell microenvironment, the intracellular mechanical stress also destroyed the cell cytoskeleton, as observed by transmission electron microscopy (TEM) images of HepG-2 cells (Fig. 6g) and fluorescence images of cytoskeleton actin network (Fig. 6h) after incubation with AMLs, which was attributed to the ultrasound-stimulated intracellular blasting of bubbles. A 2D map of stress was also estimated using a numerical model to determine the force threshold for destroying the cell cytoskeleton (Fig. 6f).

Fig. 6.

Anticancer effects of AMLs through magnetic targeting, H₂S bubbling, as well as MRI and US dual imaging monitoring. (a) Scheme illustrating the composition of AML and its synergetic H₂S generation, tumor bombing and MRI/US dual-model imaging theranostic mechanisms. (b) Cellular morphology changes and intracellular bubble generation captured at different time points treated by different samples, in which ALs refers to ADT-loaded liposomes without encapsulating superparamagnetic nanoparticles. In vivo (c) US images and (d) MRI of mouse tumors before and after administration of different samples [60]. Copyright 2017, reproduced with permission from American Chemical Society. (e) Scheme of the equipment for magnetic field and ultrasound dual-manipulated cell membrane mechanosensing, as well as intracellular bubble blasting and intracellular redox disorder induced cell structure destruction. (f) Representative cell stress profiles simulated by 2D deformation fields. (g) TEM images of bubble-induced cell destruction after incubation for 8 h. (h) Fluorescence images of HepG-2 cells after fluorescein isothiocyanate (FITC)-phalloidin staining for F-actin characterization after different incubation times [61]. Copyright 2020, The Royal Society of Chemistry.

In addition to imaging strategies based on the inorganic components, imaging strategies based on organic compounds or polymer carriers themselves have also been actively explored for the formulation of polymeric H₂S delivery systems. Our group proposed a ratiometric photoacoustic (PA) monitored and TME initiated H₂S therapy for the detection and treatment of triple-negative breast cancer (TNBC) [35]. Ratiometric PA probe cyanine (CY) and polysulfide H₂S donor (PSD) were encapsulated in self-assembled polymeric nanoparticle formed by amphipathic block polymers (Pluronic F127). The thiol abundant TME triggered PSD to release H₂S, which exerted therapeutic effects against cancer and activated the ratiometric PA signal change of CY from 808 nm to 707 nm (Fig. 7a and b), for real-time H₂S monitoring and in vivo tumor pinpointing (Fig. 7c). CY-PSD nanoparticles showed excellent anticancer effects both in vitro and in vivo on TNBC cells and tumor-bearing mice. Pharmacological analysis revealed that CY-PSD nanoparticles exerted anticancer effects by inducing mitochondrial dysfunction and down-regulating oxidative stress. Besides utilizing small molecular compounds as imaging agents, polymers with intrinsic imaging property were also applied to establish H₂S delivery systems in our following research [64]. A PEG-grafted conjugated polymer (CP-PEG) was synthesized, which was then employed for loading polysulfide H₂S donor 2,2′-dipyridyl tetrasulfide (Pry-Ps) to prepare H₂S delivery nanoparticles (named as Pry-Ps@CP-PEG) (Fig. 7d). The wide NIR absorption cross-section of CP-PEG endowed highly efficient photothermal performance (Fig. 7h) for cancer cell killing and simultaneous NIR-II fluorescence (Fig. 7e) for tracing the tumor and monitoring the entire therapeutic process (Fig. 7g). More importantly, due to the generation of H₂S activated by GSH (Fig. 7f), Pry-Ps@CP-PEG was not only able to induce mitochondrial dysfunction of cancer cells but could also act as a nanoregulator to dramatically downregulate the level of proinflammation cytokines generated during photothermal therapy (PTT) without hindering the immune therapeutic performance of PTT.

Fig. 7.

Polysulfide-loaded polymeric H₂S delivery systems for PA or NIR-II fluorescence guided cancer therapeutics. (a) Mechanism of H₂S generation from PSD with GSH and response process of CY to H₂S. (b) PA signal changes for CY nanoparticles (15 μM) at 707 and 808 nm with different concentrations of NaHS (0–40 μM) (above), as well as PA signal changes of CY-PSD nanoparticles (15 μM) at 707 and 808 nm with GSH (0–6 mM) (below). (c) PA imaging of H₂S in tumor-bearing mice before and after systemic administration of CY-PSD and CY nanoparticles [35]. Copyright 2020, Wiley-VCH. (d) Schematic illustration of the NIR-II fluorescence traced inflammation nanoregulator for dual-functional H₂S and photothermal therapy with modulated immunogenicity. (e) The emission and absorption spectra of Pry-Ps@CP-PEG. (f) GSH-activated H₂S releasing kinetics of Pry-Ps@CP-PEG (40 mg L^-1). (g) The NIR-II fluorescence imaging of 4T1-tumor-bearing mice with Pry-Ps@CP-PEG via intravenous injection (λ_ex = 808 nm), in which white circles represent the injection site of tumor cells on breast pad. (h) The thermal imaging of tumor region (white circles) [64]. Copyright 2021, Wiley-VCH.

3.5. Bacteria-associated therapy

Compared with H₂S generated by mammals, bacteria derived H₂S has been identified for centuries, although it was long perceived primarily as a byproduct of bacterial metabolism. For example, sulfate-reducing bacteria utilize a wide range of organic compounds as substrates to reduce sulfate to H₂S and generate metabolic energy [148,149]. H₂S-generating enzymes homologous with mammalian CBS, CSE, or 3-MST, at least in part, were identified in most of nonsulfur bacteria [150], and the probable physiological functions of bacteria-derived H₂S were observed recently [151]. However, the metabolic and signaling pathways of this gas in bacteria are still far from clear. The other two gasotransmitters, NO and CO, both exert bactericidal effects at relatively high concentrations [[152], [153], [154]] and have been incorporated with polymeric systems for antibacterial applications [[155], [156], [157], [158]]. However, the antibacterial property of H₂S seems not so definite as NO and CO. Indeed, high concentration of H₂S (close to millimolar level) is able to induce severe oxidative damage to bacteria [159]. But such high concentration of H₂S would also induce cytotoxicity and inflammation to mammals [8]. Therefore, H₂S is generally not considered to be potential antibacterial agent like NO and CO. In contrast, H₂S could act as cytoprotective agent to protect bacteria from harmful oxidative stress imposed by antibiotics [150]. One major mechanism is H₂S-mediated sequestration of free iron to prevent the Fenton reaction that generates toxic hydroxyl radicals [160]. Besides, although garlic-derived diallyl polysulfanes, the natural H₂S donors, are known to exhibit broad spectrum antimicrobial activity [161], there is growing evidence that the property might not derived from the generation of H₂S, but the hydrogen polysulfanes (e.g., H₂S₂ and H₂S₃) and other RSSs [162,163].

Nevertheless, recent advances about the interaction between H₂S and bacteria has led to the revaluation of its potential in antibacterial therapy. Though the direct antibacterial effect of H₂S is limited, H₂S is able to relieve inflammation induced by acute bacterial infection [164], as well as lipopolysaccharide of gram-negative bacteria [165], which indicates the potential role of H₂S-releasing agents in the treatment of bacterial infections through anti-inflammatory pathways. In addition, H₂S could exhibit excellent synergistic therapeutic effects towards hard-to-heal infected wounds. Recently, Du et al. developed a H₂S delivery polymersome, which was utilized in the form of spray for treating bacteria-infected diabetic wounds (Fig. 8a) [66]. The polymersome was prepared through the self-assembly of diblock copolymer containing hydrophobic PCL block to form the membrane and poly[lysine-stat-(S-aroylthiooxime)] [P(Lys-stat-SATO)] to form the corona. In the presence of cysteine, the polymersome could generated H₂S gradually for up to 12 h (Fig. 8b). Intracellular generation of H₂S in normal human epidermal keratinocytes (NHEK) cells was also observed using a H₂S fluorescent probe (Fig. 8c). Due to the positively charged lysine amino groups in the outer corona of the polymersome, it possessed intrinsic antibacterial property through the electrostatic interaction with negatively charged bacterial membranes, showing potent bactericidal effects on both gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus) in vitro (Fig. 8d), as well as infected diabetic wounds (Fig. 8g). And the slow and sustained release of H₂S could accelerate the healing of diabetic wounds (Fig. 8e and f).

Fig. 8.

H₂S delivery polymersome for infected diabetic wound healing. (a) Schematic illustration of the formation and mechanism of action of the H₂S delivery polymersome against infected diabetic wound. (b) H₂S releasing profiles of polymersome in the presence and absence of cystine. (c) Exogenous H₂S levels in NHEK cells determined using a fluorescent probe. (d) Live/Dead staining analysis of bacteria after the treatment of polymersome. (e-f) In vivo wound healing rates of S. aureus-infected diabetic wound site treated with polymersome + Cystine and other control groups. (g) Bacterial colonies obtained from infected wounds on Day 10 treated with different groups [66]. Copyright 2021, American Chemical Society.

Biofilms are heterogeneous communities of bacteria encapsulated in a self-produced polymer matrix, providing potent defense against the killing effects of antibiotics and host immune systems [166]. Recent studies pointed out that H₂S may act as an important biofilm disruptor at proper concentration. H₂S delivery hydrogel developed by Matson et al. showed biofilm dispersal efficacy on S. aureus [67]. This hydrogel was formed through the self-assembly of SATO-functionalized amphiphilic dipeptides (S-FE). The pretreating of S-FE hydrogel before biofilm formation led to 57% reduction of biofilm biomass, more effective than C-FE hydrogel (39% reduction), the control hydrogel bearing oxime group but incapable of H₂S release. S-FE hydrogel treatment on established biofilms also led to 8% reduction in biomass, compared with a 2% decrease for the C-FE. As far as we know, this work provided the first example of the application of H₂S-releasing materials in anti-biofilm research, but the underlying mechanism is still unknown. NO, another gasotransmitter, is able to induce biofilm dispersal by stimulating bacterial phosphodiesterase activity to decrease the intracellular levels of cyclic-diguanylate-guanosine monophosphate (c-di-GMP), the intracellular second messenger of bacteria responsible for regulating the formation of biofilms [167,168]. In mammalian systems, H₂S is able to enhance the production of NO by inducting the expression of endothelial NO synthase and stimulating its activity [169,170]. Whether H₂S-induced biofilm dispersal involves the pathway of promoting NO generation in bacterial cells is still unclear, but it deserves further investigation.

Gut microbiota is an important source of H₂S in human body. H₂S in gastrointestinal tract is able to influence many physiological and pathophysiological processes [171]. On the one hand, the bacteria-derived H₂S acts as a metabolic fuel of epithelial cells, and is involved in several aspects of mucosal defense and repair [172]. On the other hand, delivery of H₂S into the colon could influence the state of microbiota and promote harmonious coexistence of the bacteria with the gastrointestinal mucosa, which showed the potential in the treatment of colonic diseases, such as inflammatory bowel disease and colorectal cancer [173,174]. In addition, the research on gut microbiota has revealed that bacteria in gastrointestinal tract have a profound impact on human health. Imbalance of gut microbiota will not only induce gastrointestinal tract disorder, but also lead to a number of diseases residing in organs far from the gut, such as obesity, allergy, type 1 diabetes, and autism [175]. Thus, delivery of exogenous H₂S through polymeric carriers to regulate gut microbiota is an approach worth exploring.

3.6. Therapies for other diseases

Like myocardial ischemia we mentioned above, ischemic diseases such as limb ischemia and ischemic stroke, are usually associated with hostile inflammatory response in the ischemic area caused by the production of large amounts of reactive oxygen species [176]. If not rescued timely, they may cause serious injuries and even death. H₂S has shown the abilities to relieve oxidative stress [177], promote angiogenesis [16], and inhibit cellular apoptosis [178], which are benefit for treating ischemia. In an attempt to treat limb ischemia, Sung et al. prepared DATS loaded H₂S releasing PLGA microparticles (DATS@MPs) through an oil-in-water single emulsion method [68]. Compared with free DATS, DATS@MPs enabled prolonged intracellular H₂S generation, favoring long-lasting therapeutic effects. After intramuscular injection to the ischemic limb of mouse model, DATS@MPs exhibited blood perfusion compared with free DATS, which was highly associated with the increase of capillary and arteriole densities at the ischemic limb. Meanwhile, the DATS@MPs group showed the lowest oxidative stress level at the ischemic tissues, illustrating the cytoprotective effect enabled by sustained H₂S release.

Diabetes is characterized by disorders of glucose metabolism, which may induce serious complications, posing a major social health threat. In recent years, growing evidences have indicated that H₂S homeostasis plays an important role in diabetes [179]. H₂S could provide a protective effect on islet β cells from oxidative stress and elevated inflammations, to maintain the regular function of islet β cells [20]. In addition, H₂S could relieve hyperglycemic endothelial dysfunction, which may be of great significance to maintain diabetic blood vessel patency and prevent the development of diabetic complications [179]. Polymeric nanomedicines open a promising option for the management of diabetes [180]. Aimed at the high cellular oxidative stress during diabetes, administrating antioxidants to regulate the redox balance have proven to be an effective approach [181]. Thus, we suppose that delivering H₂S by means of polymeric carriers is worth exploring for alleviating diabetes and its complications. Noteworthy, the role of H₂S in diabetes is complicate, and H₂S could also affect insulin secretion of islet β cells by opening K_ATP channel [182]. Therefore, the role of H₂S at different stages of diabetes should be further explored to provide guidance for exogenous H₂S delivery.

In recent years, H₂S also showed therapeutic effect on neurodegenerative diseases, such as Alzheimer's disease. Neurodegenerative diseases are highly associated with oxidative stress [183], and delivering antioxidants through polymeric nanomedicines is explored as a method for treating neurodegenerative diseases [184]. As mentioned above, H₂S could prevent oxidative stress through various pathways. In addition, more recent studies revealed that H₂S showed neuroprotective effects in Alzheimer's disease by inhibiting the hyperphosphorylation of Tau protein [185], while the regulation of Tau pathology has shown great potentials on the treatment of Alzheimer's disease [186]. Therefore, polymeric H₂S delivery systems deserve to be explored for treatment of neurodegenerative diseases represented by Alzheimer's disease. Noteworthy, one important property they should be endowed is blood-brain barrier penetrability.

4. Conclusion and future prospects

The past two decades have witnessed the rise of studies on the physiological effects of H₂S, opening a promising option for the treatment of various human diseases. Since the biological effects of H₂S are highly associated with its concentrations, there is urgently needed to develop strategies for elaborately regulated H₂S delivery. To meet the requirements of precision medicines, intelligent polymeric H₂S delivery systems capable of targeting the lesions specifically, responding to pathological microenvironments smartly and employing multi-model imaging technologies for real-time theranostics are actively explored, showing excellent accuracy and opening a new option for the next generation of H₂S therapy. Here, we provide a comprehensive overview of intelligent polymeric H₂S delivery systems and their therapeutic potentials towards cardiovascular diseases, inflammatory diseases, tissue defect, cancers, bacteria-associated diseases, ischemic diseases, diabetes and neurodegenerative diseases.

In recent years, the development of advanced bioimaging technology provides great opportunities for establishing intelligent polymeric H₂S delivery systems that integrate diagnosis, treatment, and real-time monitoring of various chronic diseases. We hope that visualized H₂S therapy could be realized by deep deconstruction of the pathological microenvironments, rational design of polymer architectures and elaborate integration of specific functional moieties. However, the development of intelligent polymeric H₂S delivery systems is still in the infancy stage, and there is still a long way to achieve their clinical translation. Accurate diagnosis depends on the discovery and analysis of more relevant biomarkers. Effective treatment also requires a deeper study on the dose-dependent biological effects of H₂S. We are convinced that the incorporation of precision medicines and H₂S therapy would promote the revolutionary development of the next generation of therapies.

In addition to their potential in precision medicines, some issues are also worth noting. The understanding of H₂S biology is constantly being updated in recent years, which may lead to breakthroughs in the treatment of some chronic diseases, such as neurodegenerative diseases, diabetes and imbalance of gut microbiota. The further expansion of H₂S therapy also depends on the development of H₂S donors with improved biosafety and optimized activating mechanisms. In addition, since other RSSs (such as persulfide and H₂S_n [187]) and their selenium analogue such as H₂Se [188] have also been proven to exert specific regulating effects in various physiological processes, precision medicine of these RSSs therapies is worthy of further exploration.

CRediT authorship contribution statement

Fan Rong and Tengjiao Wang contributed equally. Fan Rong: Writing - Original Draft, Visualization. Tengjiao Wang: Conceptualization, Investigation, Writing - Original Draft, Funding acquisition. Qian Zhou: Writing - Review & Editing. Haowei Peng: Writing - Original Draft. Jingtian Yang: Writing - Review & Editing. Quli Fan: Supervision, Peng Li: Conceptualization, Supervision, Funding acquisition, Writing - Review & Editing, Project Administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.