Stimuli-Responsive Poly(Aspartamide) Derivatives and Their Applications As Drug Carriers

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International Journal of Molecular Sciences

Review Stimuli-Responsive Poly(aspartamide) Derivatives and Their Applications as Drug Carriers

Guangyan Zhang 1,2,* , Hui Yi 1 and Chenhui Bao 1

1 School of Materials and Chemical Engineering, Hubei University of Technology, Wuhan 430068, China; [email protected] (H.Y.); [email protected] (C.B.) 2 Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, Wuhan 430068, China * Correspondence: [email protected]

Abstract: Poly(aspartamide) derivatives, one kind of amino acid-based polymers with excellent biocompatibility and biodegradability, meet the key requirements for application in various areas of biomedicine. Poly(aspartamide) derivatives with stimuli-responsiveness can usually respond to external stimuli to change their chemical or physical properties. Using external stimuli such as temperature and pH as switches, these smart poly(aspartamide) derivatives can be used for convenient drug loading and controlled release. Here, we review the synthesis strategies for preparing these stimuli-responsive poly(aspartamide) derivatives and the latest developments in their applications as drug carriers.

Keywords: poly(aspartamide) derivatives; stimuli-responsive; drug carrier; nanoparticles; hydrogel; polymer-drug conjugate; drug-loading; controlled drug release

Citation: Zhang, G.; Yi, H.; Bao, C. Stimuli-Responsive Poly(aspartamide) Derivatives and 1. Introduction Their Applications as Drug Carriers. Though the carbon-carbon backbone is conducive to the stability of polymers, it also Int. J. Mol. Sci. 2021, 22, 8817. limits the applications of these polymers in biomedical fields due to its low biocompat- http://doi.org/10.3390/ijms22168817 ibility and non-biodegradability. In the past decades, the interest in amino acid-based biodegradable polymers has increased significantly in biomedicine due to their good Academic Editors: Ádám Juhász and biocompatibility, biodegradability and non-toxicity of their degradation products. For Edit Csapó example, poly(glutamic acid) (PGA) and PGA-based polymers have been widely applied for controlled release of peptide, protein and anti-tumor drugs [1]; polylysine (PLL) and Received: 29 July 2021 its derivatives have been extensively investigated in gene delivery systems [2]. Moreover, Accepted: 13 August 2021 Published: 16 August 2021 other amino-acid containing degradable polymers have also been studied in tissue engineering and biomedical imaging (e.g., magnetic resonance imaging and fluorescence imaging). Poly(aspartic acid)-based polymers, whose backbone is composed of aspartic acid, are Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in one of the most intensively studied amino acid-based polymers in drug [3,4]/gene [5–7] de- published maps and institutional affil- livery systems and other biomedical applications [8] due to their simple synthesis method, iations. excellent biocompatibility and biodegradability. The synthetic strategies of poly(aspartic acid)-based polymers mainly include the aminolysis reaction of poly(succinimide) (PSI) (Figure1a) and the ring-opening polymerization (ROP) of benzyl- L-aspartate N-carboxyanhydride (BLA-NCA) (Figure1b). For side chain-modified poly(aspartic acid)- based polymers, they can generally be prepared by the aminolysis reaction of PSI. For Copyright: © 2021 by the authors. example, a side chain-modified poly(aspartamide) (PASPAm) derivative, α,β-poly(N- Licensee MDPI, Basel, Switzerland. D L This article is an open access article 2-hydroxyethyl)- , -aspartamide (PHEA), can be obtained by the aminolysis reaction distributed under the terms and between ethanolamine and PSI, and has been further modified and intensively studied as conditions of the Creative Commons scaffolds [9], siRNA delivery systems [10,11] and drug carriers (e.g., hydrogel, nanoparti- Attribution (CC BY) license (https:// cles) [12–16]. For the block copolymers containing poly(aspartic acid)-based segment, they creativecommons.org/licenses/by/ usually can be synthesized by ROP of BLA-NCA. For instance, NK105 polymer, which is 4.0/).

Int. J. Mol. Sci. 2021, 22, 8817. https://doi.org/10.3390/ijms22168817 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18

block as the hydrophobic segment in which 50% carboxylic groups of PASPAm were reacted with 4-phenyl-1-butanol by esterification reaction [17,18], has been used as delivery system in a paclitaxel-incorporating micellar nanoparticle anti-cancer formulation (NK105) that was approved for clinical trials (phase III) [19,20]. Besides NK105, poly(aspartic acid)- Int. J. Mol. Sci. 2021, 22, 8817 based polymers were also applied in two other formulations, NK911 [21] and NC2-6300 of 18 [22,23]. Among the above three clinical formulations, NC-6300 that is an epirubicin (EPI) conjugated PEG-block-PASPAm derived copolymer containing acid-labile hydrazone link- composedages, is the ofonly a poly(ethyleneone with stimuli glycol)-responsiveness. (PEG) block Results as the showed hydrophilic that NC segment-6300 exhibited and modi- a ﬁedstronger poly(aspartic anti-tumor acid)-based effect and block lower as cardioto the hydrophobicxicity compared segment to in free which EPI, 50% which carboxylic may be groupsdue to its of pH PASPAm responsiveness. were reacted with 4-phenyl-1-butanol by esteriﬁcation reaction [17,18], has beenBecause used stimuli as delivery-responsive system polymers in a paclitaxel-incorporating can respond to the micellarchanges nanoparticleof environmental anti- cancerfactors formulation(e.g., pH, temperature) (NK105) that by was self approved-changing for either clinical their trials physical (phase properties III) [19,20,]. chemical Besides NK105,structures poly(aspartic, or both, they acid)-based exhibit attractive polymers prospects were also in applieda variety in of two fields other such formulations, as sensors, NK911energy conversion [21] and NC-6300 and drug/gene [22,23]. Amongdelivery thesystems above [24] three. Therefore, clinical formulations,various stimuli NC-6300-respon- thatsive isPASPAm an epirubicin derivatives (EPI) conjugatedsuch as NC PEG-block-PASPAm-6300 have been designed derived and copolymer synthesized containing over the acid-labilelast decades, hydrazone and their linkages,potentialis applications the only one in with different stimuli-responsiveness. fields were also investigated Results showed exten- thatsively. NC-6300 In this exhibitedreview, PASPAm a stronger derivatives anti-tumor are effect defined and as lower polymers cardiotoxicity composed compared wholly toor freepartly EPI, of whichaspartamide may be units due toas itsshown pH responsiveness.in Figure 1.

FigureFigure 1. The synthetic pathways of PA PASPAm-basedSPAm-based ((aa)) graftgraft copolymerscopolymers andand ((bb)) blockblock copolymers.copolymers.

BecauseAlthough stimuli-responsive some papers have polymersreviewed canthe synthesis respond toand the applications changes of of environmental poly(aspartic factorsacid) (PASP) (e.g., [25] pH, and temperature) PSI [26] derivatives, by self-changing there is eitherno comprehensive their physical review properties, summarizing chem- icalthe recent structures, progress or both,of “stimuli they-responsive exhibit attractive PASPAm prospects derivatives”. in a varietyIn this paper, of fields we will such pro- as sensors,vide an overview energy conversion of the up-to and-date drug/gene developments delivery on stimuli systems-responsive [24]. Therefore, PASPAm various deriva- stimuli-responsivetives and their applications PASPAm as derivativesdrug carriers. such First, as the NC-6300 synthesis have strat beenegies designed of stimuli and-respon- syn- thesizedsive PASPAm over thederivatives last decades, will be and reviewed their potential by the type applications of triggers. in Next, different the applications fields were alsoof stimuli investigated-responsive extensively. PASPAm In derivatives this review, for PASPAm drug delivery derivatives will are be definedintroduced. as polymers Finally, composedthe further whollyperspectives or partly of stimuli of aspartamide-responsive units PASPAm as shown de inrivatives Figure 1will. be discussed. Although some papers have reviewed the synthesis and applications of poly(aspartic acid) (PASP) [25] and PSI [26] derivatives, there is no comprehensive review summarizing the recent progress of “stimuli-responsive PASPAm derivatives”. In this paper, we will provide an overview of the up-to-date developments on stimuli-responsive PAS- PAm derivatives and their applications as drug carriers. First, the synthesis strategies of stimuli-responsive PASPAm derivatives will be reviewed by the type of triggers. Next, the applications of stimuli-responsive PASPAm derivatives for drug delivery will be in- Int. J. Mol. Sci. 2021, 22, 8817 3 of 18

troduced. Finally, the further perspectives of stimuli-responsive PASPAm derivatives will be discussed.

2. Synthesis 2.1. Temperature-Responsive Poly(aspartamide) Derivatives Temperature is an important stimulus for stimuli-responsive polymers. Poly(N- isopropylacrylamide) (PNIPAAm) is one of the most investigated temperature-responsive polymers with a lower critical solution temperature (LCST) at around 32 ◦C. As is well known, the temperature-responsive behavior of PNIPAAm is ascribed to its N-isopropylamide pendants. Therefore, N-isopropylamide and various functional groups with similar structures were introduced for preparing temperature-responsive PASPAm derivatives. For instance, two different temperature-responsive PASPAm derivatives, PAIPAHA and PAA- TS, were prepared by the following procedures: (1) First, a certain amount of isopropylamine was reacted with PSI, and then (2) alkanolamines [27] or NaOH [28] were further used to open the remaining succinimide rings to obtain temperature responsiveness. Besides N-isopropylamide pendants, diisopropylamide [29] and isopropylethylene- diamide [30,31] pendants were also introduced for the design of temperature-responsive PASPAm derivatives. In fact, in the absence of isopropylamide or diisopropylamide pendants, PASPAm derivatives containing alkanolamides moieties also can possess temperature responsiveness. In 2003, a series of temperature-responsive PASPAm(C5OH/C6OH) were synthesized via the aminolysis reaction of PSI with a mixture of 5-aminopentanol (NH2C5OH) and 6-aminohexanol (NH2C6OH) by Kobayashi’s group [32]. Interestingly, the reaction product of PSI with NH2C5OH, denoted as PHPA, was completely soluble in water; while the reaction product of PSI with NH2C6OH was insoluble in water (test temperature: 0–100 ◦C). Since then, more and more temperature-responsive PASPAm derivatives containing alkanolamides moieties were designed and synthesized [33–35]. For instance, adopting the similar strategy of Kobayashi’s group, Chu et al. prepared a new family of ◦ temperature-responsive PASPAm(C4OH/C6OH) with an LCST ranging from 28 to 53 C using the mixture of 4-aminobutanol (NH2C4OH)/NH2C6OH instead of the mixture of NH2C5OH/NH2C6OH [36]. In 2014, phenethyl alcohol (phe) was grafted to the pentanolamide pendants of PHPA, and the obtained ﬁve phe-g-PHPA polymers all exhibited temperature responsiveness. The LCST of phe-g-PHPA can be tuned by adjusting the per- centage of grafted phe moieties, and has a negative linear correlation with the grafting ratio of phe [37]. In 2015, a certain amount of 2-azidoethylamine (Az) was used for aminolysis reaction with PSI, and then excess NH2C5OH was further utilized to open the residual succinimide rings to prepare azide-functional PASPAm derivative P(Asp-Az)x-HPA. Two P(Asp-Az)x-HPA polymers showed temperature-responsive behaviors in aqueous solution, and the molar ratio of Az/NH2C5OH in these two P(Asp-Az)x-HPA polymers was 39/61 and 56/44, respectively [38]. Additionally, the LCST of P(Asp-Az)x-HPA-based polymers can be further adjusted by grafting hydrophobic moieties, such as cinnamoyl group [33]. Moreover, ether oxygen also plays an important role for the design of temperature- responsive polymers. For example, oligo(ethylene glycol) (OEG) containing ether oxygen was usually introduced for preparing temperature-responsive polymers, such as OEG methacrylate based polymers [39,40] and polypeptides bearing OEG pendants [41,42]. In 2021, linear alkyl ether-type amine compounds, which contain one ether oxygen and vary- ing carbon atoms, were successfully used for the synthesis of temperature-responsive PAS- PAm derivatives bearing alkyl ether-type pendants [43]. The composition of temperature- responsive PASPAm derivatives and their LCSTs are summarized in Table1. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEERTable REVIEW 1. A summary of temperature-responsive PASPAm derivatives. 4 of 18

Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW 44 ofof 1818 Int.Int. J. J.Mol. Mol. Sci. Sci. 2021 2021, 22, 22, x, FORx FOR PEER PEER REVIEW REVIEW 4 4of of 18 18 Int.Temperature J. Mol. Sci. Responsive2021, 22, 8817 PAS- Composition of Side Chain 4 of 18 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW R1/R2 LCST (°C) Ref4 of 18 Int. J. Mol.PAm Sci. Derivatives2021, 22, x FOR PEERTable REVIEW 1. A summarR1 y of temperature-responsiveR2 PASPAm derivatives. 4 of 18

Table 1. A summary of temperature-responsive PASPAm derivatives. TemperaturePAIPAHA Responsive PAS- Table 1. A summarCompositiony of temperature of Side-responsive ChainNH -(CH 2PA)6OHSP Am derivatives55/45. 30 [27] Int. J. Mol. Sci. 2021, 22, x FOR PEERTable TableREVIEW 1. 1. A A summar summary yof of temperature temperature-responsive-responsive PA PASPSPAmAm derivatives derivativesR1/R. 2 . LCST (°C) Ref4 of 18 TemperaturePAm Derivatives Responsive PAS- R1 Composition of Side Chain R2 TableTable 1. 1. AA summar summaryy of of temperature temperature-responsive-responsive PA PASPAmSPAm derivatives derivatives.R1/R. 2 LCST (°C) Ref TemperatureTemperaturePAm Derivatives ResponsiveResponsive PAPAS-S- Table 1. A summarR1 CompositionCompositiony of temperature ofof SideSide- responsive ChainChain R2 PASPAm derivatives. TemperatureTemperaturePAIPAPA Responsive Responsive PA PAS-S- CompositionComposition of of Side Side Chain ChainNH -(CH 2)3OH R55/451/R2 LCST40 ( °C) Ref[27] PAIPAHA NH-(CH2)6OH R55/451/R2 LCST30 (°C) [27]Ref TemperaturePPAmAm DerivativesDerivatives Responsive PAS- RR11 Composition of Side Chain RR22 R1/R2 LCST (°C) Ref PAm Derivatives R1 1 Composition of Side Chain R2 2 TemperaturePAm Derivatives Responsive R R2 6 R1/R2 LCST a(°C◦ ) Ref TemperaturePAIPAHA Responsive PAS- Composition of Side ChainNH-(CH ) OH 55/4569/31R1/R 2 LCST3830 a ( C) [27]Ref PASPAmPAm Derivatives Derivatives R1 R2 R1/R2 LCST (°C) Ref Table 1. A summarR1 y of temperature-responsiveR 2PASPAm derivatives. PAmPAIPAHAPAIPAHA Derivatives R1 NHNH--(CH(CHR22 2))66OHOH 55/4555/45 3030 a [27][27] PAIPAHAPAIPAPAPAIPAHA NHNH-(CH-(CH2)362OH)6OH 55/4573/2755/45 404030 30 [27][27] a TemperaturePAIPAHAPAIPAPA Responsive PAS- Composition of Side ChainNH- (CH2)36OH 55/4574/26 454030 [27] PAIPAHAPAIPAHA NHNH-(CH-(CH2)6)OHOH 55/45 55/45 30 30a [27] [27] 2 6 69/31R1/R2 LCST38 a( °C) Ref PAmPAIPAPAPAIPAPAPAA Derivatives-TS R1 NHNH--(CH(CHOHR22 2) )33OHOH 55/4555/4575/25 474040 [27][27][28] PAIPAPAPAIPAPA NHNH-(CH-(CH2)32OH)3OH 55/4555/45 4040a [27][27] 73/2769/31 4038 a PAIPAPA NH-(CH2)3OH 55/4577/23 4940 [27] aa PAIPAPA NH-(CH2)3OH 69/3174/2673/2755/45 38454040 aa [27] PAIPAHAPAIPAPA NHNH-(CH-(CH2)26)OH3OH 69/3178/2255/4569/31 55/45 3856303840 a [27] [27] 74/26 45 aa PAA-TS OH 73/2775/2573/2769/3180/20 4047403862 aa a [28] 73/2773/27 4040 a a 69/3169/31 38 38 aa PAA-TS OH 74/2677/2375/2574/2673/27 4549474540 a a [28] PAIPAPA NH-(CH2)3OH 74/2655/4574/2673/27 4540 4045 a a [27] 73/27 40 aaa PAAPAAPSI---TSTS OHOH- 75/2578/2277/2375/2574/2669/31 475649474534 a aa [28][28][28] PAAPAA-TS-TS OHOH 75/2575/2574/26 47 4547 a [28][28] 74/26 45 aaa a PAAPAA-TS-TS OHOH 77/2380/2078/2277/2375/2569/3175/25 496256494738 47 a a [28][28 ] 77/2377/23 4949 a a PAA-TS OH 80/2075/2577/23 6247 49 aa [28] 78/2278/2277/2373/27 56564940 a aa 78/2278/2278/22 56 5656 aa mPEGPSI-hyd-TS-PDAHy - 80/(9)2069/3177/23 344940 aa a [28][29] 80/2080/2078/2274/2680/20 62625645 62 a a 80/2080/20 6262 a PSI-TS NH--NH 2 69/3178/22 3456 a [28] PAA-TS OH 80/2075/25 6247 [28] PSI-TS - 69/3180/20 3462 aa a [28] PSIPSI-TSPSI-TS-TS - - 69/3177/2357/4369/31 69/31 344924 3434 a a [28][[28]28 ] a mPEG-PSIhyd-TS-PDAHy - 80/(9)2069/31 3440 a [29][28] 78/2258/42 5630 a PolyAspAm(HA/NIPEDA)mPEG-PSIhyd-TS-PDAHy NHNH-(CH--NH 2)5CH2 3 80/(9)2069/31 3440 [29][30][28] 59/41 39a 80/20 62 mmPEGPEG--hydhyd--PDAHyPDAHy NH-NH2 80/(9)2080/(9)2057/43 402440 [29][29] mmPEG-hyd-PDAHymPEGPEG-hyd-hyd-PDAHy-PDAHy 80/(9)2062/3880/(9)2080/(9)20 4052 4040 [29] [[29]29 ] NH-NH2 a mPEG-PSIhyd-TS-PDAHy NH--NH 2 80/(9)2057/4369/31 342440 [29][28] NHNHNH-NH-NH-NH 22 58/4257/43 3022 PolyAspAm(HA/NIPEDA)mPEG-hyd-PDAHy NH-(CH2)5CH3 80/(9)20 40 [30][29] NH-NH2 57/4359/4158/4257/43 24393024 PolyAspAm(HA/NIPEDA) NH-(CH2)5CH2 3 57/4359/4157/4357/43 2436 2424 [30] PolyAspAm(OA/NIPEDA) NHNH-(CH-NH2)7CH 3 [30] 58/4262/3859/4158/4257/4358/42 3052393024 30 PolyAspAm(HA/NIPEDA)PolyAspAm(HA/NIPEDA) NHNH-(CH-(CH2)52CH)5CH3 3 61.5/39.558/4258/42 304330 [30][30 ] PolyAspAm(HA/NIPEDA) NH-(CH2)5CH3 57/4359/41 24 39 [30] PolyAspAm(HA/NIPEDA)mPEG-hyd-PDAHy NH-(CH2)5CH3 80/(9)2059/4157/4359/4162/3858/42 39223952304940 [29][30] PolyAspAm(HA/NIPEDA) NH-(CH2)5CH3 59/4162/38 5239 [30] NH-NH2 62/3857/4358/42 522230 PolyAspAm(HA/NIPEDA) NH-(CH2)5CH3 59/4162/3857/43 36523922 22 [30] PolyAspAm(OA/NIPEDA) NH-(CH2)7CH3 59/4162/38 3952 [30] 2 11 3 61.5/39.557/4359/4157/4362/3859/41 224336225224 36 PolyAspAm(OA/NIPEDA)PolyAspAm(LA/NIPEDA)PolyAspAm(OA/NIPEDA) NHNH-(CH--(CH(CH22))1127CH)CH7CH33 3 57/4361/3957/43 223722 [30,31][30][30 ] 61.5/39.562/3860.5/39.5 4352 43 59/4162/3859/4157/4364/3658/42 364936225030 PolyAspAm(OA/NIPEDA)PolyAspAm(OA/NIPEDA)PolyAspAm(HA/NIPEDA) NHNH--(CH(CH22))775CHCH33 59/4162/38 4936 [30][30] PolyAspAm(OA/NIPEDA)PolyAspAm(OA/NIPEDA) NHNH-(CH-(CH2)72CH)7CH3 3 61.5/39.562/3857/43 434922 [30][30] 61.5/39.559/4150/5059/41 2243362339 22 PolyAspAm(OA/NIPEDA) NH-(CH2)7CH3 59/4161.5/39.5 3643 [30] PolyAspAm(LA/NIPEDA)PolyAspAm(LA/NIPEDA) NHNH-(CH-(CH2)211)CH11CH3 3 62/3861/3959/4161/39 493722 37 [30,31][30,31] PolyAspAm(OA/NIPEDA) NH-(CH2)7CH3 61.5/39.562/3860/4062/38 4943295249 [30] PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH 64/36 50 [32] PolyAspAm(LA/NIPEDA)PAspAm(C5OH/C6OH) NH-(CH ) OH NHNH-(CH-(CH2)11) CHOH3 61.5/39.559/4164/3661/39 22503743 [30,31][32] 59/4162/3870/3057/4359/4150/50 224937 2322 62/38 49 PolyAspAm(LA/NIPEDA)PolyAspAm(LA/NIPEDA) NHNH--(CH(CH22))1111CHCH33 61/3950/5064/3661/3959/4160/40 372350372236 29 [30,31][30,31] PolyAspAm(LA/NIPEDA)PolyAspAm(LA/NIPEDA)PAspAm(C5OH/C6OH) NH-(CH ) OHNHNH NH-(CH-(CH-(CH2)112)CH)11OHCH3 3 61/3980/2061/39 374437 [30,31][30,31][32] PolyAspAm(OA/NIPEDA) 2 5 NH-(CH2)72CH6 3 59/4170/30 22 37 [30] PolyAspAm(LA/NIPEDA) NH-(CH2)11CH3 61.5/39.564/3660/4050/5064/3661/39 502923503743 [30,31] 2 5 2 6 64/3630/7064/36 505350 PolyAspAm(LA/NIPEDA)PAspAm(C5OH/C6OH) NH-(CH ) OH NHNH--(CH(CH2))11OHCH 3 61/3980/20 37 44 [30,31][32] 50/5070/3060/4050/5064/3662/38 233729235049 PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH 50/5025/7550/5030/70 2340 5323 [32] 60/4070/3064/36 293750 PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH 80/2060/4050/5020/8059/4125/75 4429233822 40 [36] PAspAm(C5OH/C6OH)PAspAm(C5OH/C6OH) NHNH--(CH(CH22))55OHOH NHNH--(CH(CH22))66OHOH 60/40 29 [32][32] PAspAm(C5OH/C6OH)PAspAm(C4OH/C6OH)PAspAm(C5OH/C6OH) NHNH-(CHNH-(CH-(CH22))452OH)5OH NHNH NH-(CH-(CH-(CH2)262OH))66OH 80/2050/5020/80 4423 38 [32][[32]36 ] PolyAspAm(LA/NIPEDA) NH-(CH2)11CH3 70/3030/7070/3060/4015/8561/39 3753372932 [30,31] PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH 70/3015/85 3237 [32] 80/2030/7060/40 445329 PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH 25/7580/2070/3010/9064/3610/90 4044372850 28 [32] 70/3080/20 3744 PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH (30/7020/8030/7025/7580/20250/50)/100 53385340445423 [36] 80/20(2)/10030/70 44 5453 PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH (25/7515/8525/7520/8030/70660/40)/100 403240385329 [36] PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH 25/75 40 [32][37] 2 4 2 6 30/70(6)/100 53 32 PAspAm(C4OH/C6OH)PAspAm(C4OH/C6OH)phe-g-PHPA NHNH--(CH(CH2))4OHOH NHNHNH-(CH--(CH(CH22))65OHOH) OH 20/8010/9015/8520/8025/7570/30 382832384037 [36][36] PAspAm(C4OH/C6OH)PAspAm(C4OH/C6OH)phe-g-PHPA NHNH-(CH-(CH2)42OH)4OH NHNH--(CH(CH-(CH2))262OH)56OH (1120/80)20/80/100 382038 [36][37][36] (15/85210/9025/75)(11)/100/100 32542840 20 PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH (1215/8520/8080/20(12)/100)15/85/100 32381444 1432 [36] PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH 20/80 38 [36] ((1310/90610/90215/8530/70)(13)/100)//100100 28322854539 9 15/8510/90 3228 phe-g-PHPA NH-(CH2)5OH (((1126))/)/100/100100 542032 b [37] (0/22.2/30.3)/100(210/9025/75()2/100)/100 35.754284054 phe-g-PHPA NH-(CH2)5OH (1110/90)/100 2028 [37] PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH (0/26.6/28.3)/100(((1266220/80))/)/100/100100 31.53214325438 b [36] (2()6/100)/100 5432 phephe--gg--PHPAPHPA NHNH--(CH(CH22))55OHOH ((11(131211615/85))//100100 201420329 b [37][37] phephe-g-gPHPA-PHPA NHNH-(CH-(CH2)52OH)5OH (0/37.6/27.3)/100(11(11)/100)/100 22.52020 b [37][37] (0/22.2/30.3)/100(6)/100 35.732 b phe-g-PHPA NH-(CH2)5OH (0/22.2/30.3)/100(0/39.6/19.0)/100((1213121110/90))//100100 35.729.5141420289 [37] (12(12)/100)/100 1414 b phe-g-PHPA NH-(CH2)5OH (0/26.6/28.3)/100(11)/100 31.520 b [37] (0/26.6/28.3)/100(0/22.2/30.3)/100(0/53.6/12.0)/100((13(13122))//100100 31.535.745.8145499 (13)/100 9 b (0/37.6/27.3)/100(12)/100 22.514 bb (0/22.2/30.3)/100(0/37.6/27.3)/100(0/26.6/28.3)/100(0/22.2/30.3)/100((136))//100100 35.722.531.535.7329 b b b [34] Phe/DEAEPhe/DEAE-g-PHPA-g-PHPA NHNH-(CH-(CH2)25)OH5OH (0/22.2/30.3)/100(5/34.6/12.7)/100(0/22.2/30.3)/100 35.72835.7 [34] (0/39.6/19.0)/100(13)/100 9 29.5 bb phe-g-PHPA NH-(CH2)5OH (0/26.6/28.3)/100(0/39.6/19.0)/100(0/37.6/27.3)/100(0/26.6/28.3)/100(0/22.2/30.3)/100(11)/100 31.522.531.535.729.520 b b [37] (0/26.6/28.3)/100(0/26.6/28.3)/100 31.531.5 b (0/22.2/30.3)/100 35.7 bb (0/37.6/27.3)/100(0/53.6/12.0)/100(0/39.6/19.0)/100(0/37.6/27.3)/100(0/26.6/28.3)/100(0/53.6/12.0)/100(12)/100 22.522.531.545.829.514 45.8 b b (0/37.6/27.3)/100(0/37.6/27.3)/100 22.522.5 b (0/39.6/19.0)/100(5/34.6/12.7)/100(0/53.6/12.0)/100(0/26.6/28.3)/100 31.529.545.828 b b b Phe/DEAE-g-PHPA NH-(CH2)5OH (0/39.6/19.0)/100(0/37.6/27.3)/100(5/42.4/10.5)/100(0/39.6/19.0)/100(5/34.6/12.7)/100(13)/100 22.529.51592829.5 b [34] (0/37.6/27.3)/100 22.5b b (0/53.6/12.0)/100(5/34.6/12.7)/100 45.828 b Phe/DEAE-g-PHPA NH-(CH2)5OH (0/53.6/12.0)/100(0/39.6/19.0)/100(0/22.2/30.3)/100(0/53.6/12.0)/100(5/42.4/10.5)/100 35.745.829.51545.8 [34] (5/34.6/12.7)/100(0/39.6/19.0)/100 2829.5 bb b Phe/DEAEPhe/DEAE--gg--PHPAPHPA NHNH--(CH(CH22))55OHOH (5/34.6/12.7)/100(0/53.6/12.0)/100(0/26.6/28.3)/100 31.545.828 b b [34][34] Phe/DEAEPhe/DEAE-g-gPHPA-PHPA NHNH-(CH-(CH2)52OH)5OH (5/34.6/12.7)/100 28 [34][34] (0/53.6/12.0)/100 45.8b b Phe/DEAE-g-PHPA NH-(CH2)5OH (5/42.4/10.5)/100(5/34.6/12.7)/100(0/37.6/27.3)/100 22.51528 [34] (4)/100 32b Phe/DEAE-g-PHPA NH-(CH2)5OH (5/34.6/12.7)/100 28 b [34] (5/42.4/10.5)/100(0/39.6/19.0)/100 29.515 NB-g-PHPA NH-(CH2)5OH (7.5)/100 21 [35] (5/42.4/10.5)/100(0/53.6/12.0)/100 1545.8 bb (5/42.4/10.5)/100(5/42.4/10.5)/100(10)/100 151115 b b b Phe/DEAE-g-PHPA NH-(CH2)5OH (5/42.4/10.5)/100(5/34.6/12.7)/100(4)/100 152832 b [34] (5/42.4/10.5)/100(12)98.5/1.5 1525 b NB-g-PHPA NH-(CH2)5OH (7.5)/100(4)/100 2132 [35] NB-g-PHPA-g-mPEG NH-mPEG [35] NB-g-PHPA NH-(CH2)5OH (17)98.5/1.5(7.5)/100(10)/100(4)/100 3211218 [35] (4)/100(4)/100 3232 NH-(CH2)5OH b NBNB--gg--PHPAPHPA NHNH--(CH(CH22))55OHOH (5/42.4/10.5)/100(12)98.5/1.5(7.5)/100(7.5)/100(10)/100(4)/100 152125112132 [35][35] NBNB-g-gPHPA-PHPA NHNH-(CH-(CH2)52OH)5OH (7.5)/100(7.5)/100 2121 [35][35] (12)98.5/1.5(10)/100(4)/100 112532 NB-gNB-PHPA-g-PHPA-g-mPEG NHNH-(CH-mPEG2)5OH (7.5)/100(10)/100(10)/100 112111 [35] NB-g-PHPA NH-(CH2)5OH (17)98.5/1.5(7.5)/100 218 [35] NB-g-PHPA-g-mPEG NH-mPEG (12)98.5/1.5(12)98.5/1.5(10)/100 252511 [35] NH-(CH2)5OH (17)98.5/1.5(10)/100(12)98.5/1.5 118 25 (12)98.5/1.5(4)/100 2532 NBNB--gg--PHPAPHPA--gg--mPEGmPEG NH-(CH2)5OH NHNH--mPEGmPEG [35][35] NBNB-g-gPHPA-PHPA-g-gmPEG-mPEG NHNH-mPEG-mPEG (17)98.5/1.5(17)98.5/1.5(12)98.5/1.5 2588 [35][35] NB-gNB-PHPA-g-PHPA-g-mPEG NHNH-(CH-mPEG2)5OH (17)98.5/1.5(7.5)/100(17)98.5/1.5 218 8 [35] NHNH--(CH(CH22))55OHOH NB-g-PHPA-g-mPEG NHNH-(CH-(CH2)52OH)5OH NH-mPEG (17)98.5/1.5(10)/100 118 [35] NH-(CH2)5OH (17)98.5/1.5 8 NH-(CH2)5OH (12)98.5/1.5 25 NB-g-PHPA-g-mPEG NH-mPEG [35] (17)98.5/1.5 8 NH-(CH2)5OH

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 18

Int. J. Mol. Sci. 2021, 22, x FOR PEERTable REVIEW 1. A summary of temperature-responsive PASPAm derivatives. 4 of 18

Temperature Responsive PAS- Composition of Side Chain R1/R2 LCST (°C) Ref PAm Derivatives R1 R2 Table 1. A summary of temperature-responsive PASPAm derivatives. PAIPAHA NH-(CH2)6OH 55/45 30 [27]

Temperature Responsive PAS- Composition of Side Chain R1/R2 LCST (°C) Ref PAmPAIPAPA Derivatives R1 NH-(CHR2 2)3OH 55/45 40 [27]

a PAIPAHA NH-(CH2)6OH 55/4569/31 3830 [27] 73/27 40 a 74/26 45 a PAIPAPA NH-(CH2)3OH 55/45 40 [27] a PAA-TS OH 75/25 47 [28] 69/3177/23 3849 a 73/2778/22 4056 a 74/2680/20 4562 a PAA-TS OH 75/25 47 a [28] a PSI-TS - 77/2369/31 4934 a [28] 78/22 56 a 80/20 62 a mPEG-hyd-PDAHy 80/(9)20 40 [29] NH-NH2 a PSI-TS - 69/31 34 [28] 57/43 24 58/42 30 PolyAspAm(HA/NIPEDA) NH-(CH2)5CH3 [30] mPEG-hyd-PDAHy 80/(9)2059/41 3940 [29]

NH-NH2 62/38 52 57/43 2422 58/4259/41 3036 PolyAspAm(HA/NIPEDA)PolyAspAm(OA/NIPEDA) NH-(CH2)57CH3 [30] 61.5/39.559/41 3943

62/38 5249 57/4359/41 22 PolyAspAm(LA/NIPEDA) NH-(CH2)11CH3 59/4161/39 3637 [30,31] PolyAspAm(OA/NIPEDA) NH-(CH2)7CH3 [30] 61.5/39.564/36 4350

62/3850/50 4923 59/4160/40 2229 PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH [32] PolyAspAm(LA/NIPEDA) NH-(CH2)11CH3 61/3970/30 37 [30,31] 64/3680/20 5044 50/5030/70 2353 60/4025/75 2940 PAspAm(C5OH/C6OH) NH-(CH2)5OH NH-(CH2)6OH [32] PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH 70/3020/80 3738 [36] 80/2015/85 4432 30/7010/90 5328 (25/752)/100 4054 PAspAm(C4OH/C6OH) NH-(CH2)4OH NH-(CH2)6OH (20/806)/100 3832 [36] phe-g-PHPA NH-(CH2)5OH (1115/85)/100 3220 [37] (1210/90)/100 2814

((132))//100100 549 (0/22.2/30.3)/100(6)/100 35.732 b b phe-g-PHPA NH-(CH2)5OH (0/26.6/28.3)/100(11)/100 31.520 [37] Int. J. Mol. Sci. 2021, 22, 8817 (0/37.6/27.3)/100(12)/100 22.514 b 5 of 18

(0/39.6/19.0)/100(13)/100 29.59 (0/22.2/30.3)/100(0/53.6/12.0)/100 35.745.8 b bb Phe/DEAE-g-PHPA NH-(CH2)5OH (0/26.6/28.3)/100(5/34.6/12.7)/100 31.528 [34] Table 1. Cont. (0/37.6/27.3)/100 22.5 b

(0/39.6/19.0)/100 29.5 Composition of Side Chain b Temperature Responsive (5/42.4/10.5)/100(0/53.6/12.0)/100 45.815 ◦ R1/R2 LCST ( C) Ref PASPAm Derivatives b Phe/DEAE-g-PHPA R1 NH-(CHR22)5OH (5/34.6/12.7)/100 28 [34]

(4)/100 32 (4)/100 32 b NB-g-PHPA NH-(CH2)5OH (5/42.4/10.5)/100(7.5)/100 1521 [35][35] NB-g-PHPA NH-(CH2)5OH (7.5)/100 21 (10)/100(10)/100 11 11 (12)98.5/1.5 25 NB-g-PHPA-g-mPEG NH-mPEG (4)/100(12)98.5/1.5 32 25 [35] NB-g-PHPA-g-mPEG (17)98.5/1.5 8 [35] NB-g-PHPA NH-NH-mPEG(CH2)5OH (7.5)/100 21 [35] Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWNH -(CH2)5OH (17)98.5/1.5 8 5 of 18 (10)/100 11 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW NH-(CH2)5OH 5 of 18 (12)98.5/1.5 25 40/60 63 [33] NB-g-PHPA-g-mPEG NH-mPEG [35] P(Asp-Az)x-HPA NH-(CH2)2N3 NH-(CH2)5OH (17)98.5/1.539/61 8 58 40/60 63 [33][38 ] NH-(CH2)5OH 56/44 29 P(Asp-Az)x-HPA NH-(CH2)2N3 NH-(CH2)5OH 39/61 58 P(Asp-Az)x-HPA NH-(CH2)2N3 NH-(CH2)5OH 39/6140/60 5863 [38][33] 56/44 29 [38] P(Asp-Az)x-HPA NH-(CH2)2N3 NH-(CH2)5OH 56/4439/61 2958 (5)39/61(5)39/61 19 19 [38] (5)39/6156/44 1929 [38] P(Asp-Az)x-HPA-PEA NH-(CH2)5OH P(Asp-Az)x-HPA-PEA NH-(CH2)5OH [38] P(Asp-Az)x-HPA-PEA NH-(CH2)5OH (5)39/61(9.9)39/61 19 4 [38] (9.9)39/61 4 P(Asp-Az)x-HPA-PEA NHNH-(CH-(CH22))22NN33 NH-(CH2)5OH [38] (9.9)39/61 4 (5)39/61(5)39/61 22 22 NH-(CH2)2N3 (5)39/61 22 [38] P(Asp-Az)x-HPA-IMZ NH-(CH2)5OH (5)39/61 22 P(Asp-Az)x-HPA-IMZ NH-(CH2)5OH (9.7)39/61 10 [38] P(Asp-Az)x-HPA-IMZ NH-(CH2)5OH (9.7)39/61 10 [38] (9.7)39/61 10 P(Asp-Az)x-HPA-IMZ NH-(CH2)5OH [38] 2 2 3 NHNH-(CH-(CH22))22NN33 (9.7)39/61 10 (2.5)40/60 47 NH-(CH2)2N3 (2.5)40/60 47 (2.5)40/60(2.5)40/60 47 47 P(HPA-Az)-CA NH-(CH2)5OH [33][33 ] P(HPA-Az)-CA NH-(CH2)5OH (4.3)40/60 40

P(HPA-Az)-CA NH-(CH2)5OH (4.3)40/60 40 [33] NH-(CH2)2N3 (4.3)40/60 40

NH-(CH2)3OCH2CH3 - 100/- 54 PASP-OCx NHNH-(CH-(CH22))22NN33 [43] NH-(CH2)2OCH2CH3 - 100/- 87 NHNH-(CH-(CH22))33OCH2CH2CH3 3 - -100/ 100/-- 54 54 PASP-OCxa b [43] PASPa-OCx Determined at pH = 3.4;NH-(CH b Determined2)2OCH2CH3 at pH = 7.4 PBS. mPEG:- monomethoxy poly(ethyleneglycol). 100/- 87 [43] Determined at pH = NH3.4;- (CH Determined2)2OCH2CH at3 pH = 7.4 PBS. mPEG:- monomethoxy poly(ethyleneglycol).100/- 87 a b a DeterminedDetermined at pH at = pH 3.4; = 3.4;b DeterminedDetermined at atpH pH = =7.4 7.4 PBS. PBS. mPEG: mPEG: monomethoxymonomethoxy poly(ethyleneglycol). poly(ethyleneglycol). 2.2. pH-Responsive Poly(aspartamide) Derivatives 2.2. pHPoly(aspartic-Responsive acid)Poly(aspartamide) is a water soluble Derivatives polymer, and its conformation in aqueous solu- 2.2. pH-Responsive Poly(aspartamide) Derivatives tion canPoly(aspartic be affected acid) by pH is duea water to pH soluble-induced polymer, deprotonation and its conformation and protonation in aqueous of its carbox- solu- yliction acidcanPoly(aspartic bependants. affected Forby acid) pH example, isdue a to water thepH -αinduced soluble helical deprotonationconformation polymer, and ofand its poly(aspartic conformationprotonation acid) of inits taqueouscarbox-hat ap- pearedylicsolution acid in pendants. acidic can be solution affected For example, was by not pH theobserved due α helical to pH-induced in basicconformation solution deprotonation of[44] poly(aspartic, which and may protonation acid)be attributed that ap- of topearedits the carboxylic negative in acidic acidcharge solution pendants. repulsion was not For causedobserved example, by inthe basic the deprotonationα solutionhelical conformation[44] of, whichpendant may ofcarboxylic be poly(aspartic attributed acid groupstoacid) the negativethat in alkaline appeared charge environment in repulsion acidic solution. Thus, caused one was by strategy notthe observeddeprotonation for preparing in basic of pH solutionpendant-responsive [carboxylic44], whichPASP acidAm may derivativesgroupsbe attributed in alkaline is to to introduce theenvironment negative other charge.pH Thus,-sensitive one repulsion strategy groups caused for or preparingnon by-ionic the deprotonation groups.pH-responsive The introduction of PA pendantSPAm carboxylic acid groups in alkaline environment. Thus, one strategy for preparing pH- ofderivatives non-ionic is groups to introduce can usually other bepH used-sensitive to adjust groups the orphase non transition-ionic groups. pH valueThe introduction (pHt) [45]. responsive PASPAm derivatives is to introduce other pH-sensitive groups or non-ionic of non1--(3ionic-aminopropyl groups can) imidazoleusually be (API) used isto oneadjust of thethe mostphase comm transitiononly pHused value reagents (pHt )for [45] in-. groups. The introduction of non-ionic groups can usually be used to adjust the phase troducing1-(3-aminopropyl imidazole groups) imidazole to PASP (API) or PBLA.is one ofFor the instance, most comm Kim onlyet al. usedgrafted reagents imidazole for in- to transition pH value (pH )[45]. PEGtroducing-b-PASP imidazole copolymer groups int which to PASP 60% or carboxyl PBLA. For groups instance, were Kim reacted et al. with grafted the amineimidazole group to 1-(3-aminopropyl) imidazole (API) is one of the most commonly used reagents for ofPEG API-b- PASPforming copolymer amide linkages, in which resulting 60% carboxyl a pH -groupsresponsive were zwitterionic reacted with PA theSP amineAm deriva- group introducing imidazole groups to PASP or PBLA. For instance, Kim et al. grafted imidazole tivesof API [46] forming. In addition, amide pHlinkages,-responsiveness resulting alsoa pH can-responsive be obtained zwitterionic by substituting PASPAm the deriva-benzyl to PEG-b-PASP copolymer in which 60% carboxyl groups were reacted with the amine groupstives [46] of. PBLAIn addition, with API pH [47]-responsiveness. Another way also for introducingcan be obtained imidazole by substituting groups to synthesizethe benzyl group of API forming amide linkages, resulting a pH-responsive zwitterionic PASPAm pHgroups-responsive of PBLA PA withSP AmAPI derivatives[47]. Another is wayto directly for introducing react the imidazolesuccinimide groups rings to of synthesize PSI with derivatives [46]. In addition, pH-responsiveness also can be obtained by substituting the APIpH- responsive[48–53] or PA histamineSPAm derivatives [54] through is to aminolysis directly re reaction.act the succinimide Besides imidazole, rings of PSI tertiary with benzyl groups of PBLA with API [47]. Another way for introducing imidazole groups amineAPI [48 [34,55–53] –or58] histamine and other [54] groups through [30,59] aminolysis are also intensively reaction. Besideadopteds imidazole, for endowing tertiary pH to synthesize pH-responsive PASPAm derivatives is to directly react the succinimide responsivenessamine [34,55–58] to and PA SPotherAm groupsderivatives [30,59]. are also intensively adopted for endowing pH rings of PSI with API [48–53] or histamine [54] through aminolysis reaction. Besides Another strategy for preparing pH-responsive polymers is to introduce pH-cleavable responsivenessimidazole, tertiary to PA amineSPAm [34 derivatives,55–58] and. other groups [30,59] are also intensively adopted linkages, which mainly include hydrazone [60–62], imine [63], acetal, orthoester and 2,3- for endowingAnother strategy pH responsiveness for preparing to pH PASPAm-responsive derivatives. polymers is to introduce pH-cleavable dialkylmaleamidic amide linkages. Hydrazone linkage [64] plays an important role in the linkageAnothers, which strategy mainly forinclude preparing hydrazone pH-responsive [60–62], imine polymers [63], is acetal, to introduce orthoester pH-cleavable and 2,3- preparation of pH-responsive polymers. The most well-known pH-responsive PASPAm de- preparationdialkylmaleamidiclinkages, which of pH- mainlyresponsive amide includelinkages. polymers hydrazone Hydrazone. The most [60 linkage– well62],- imineknown [64] plays [63 pH],- acetal,responsivean important orthoester PA roleSPAm and in de-the 2,3- rivative is NC-6300, in which EPI is bound to a PEG-PASPAm block copolymer through rivativepreparationdialkylmaleamidic is NC of -pH6300,-responsive amidein which linkages. polymersEPI is Hydrazonebound. The mostto a linkagePEG well--PASPAmknown [64] plays pH block-responsive an importantcopolymer PASP role throughAm in de- the hydrazonerivative is NClinkage-6300, [23] in .which Thus, EPIEPI iscan bound be released to a PEG under-PASPAm acidic blockenvironments, copolymer such through as in lysosomes.hydrazone Doxorubicinlinkage [23]. (DOX)Thus, EPI-conjugated can be released PASPAm under derivatives acidic environments,with hydrazone such linkages as in werelysosomes. also designed Doxorubicin and prepared (DOX)-conjugated by researchers PASP [65Am–68] derivatives. Moreover, withtwo PAhydrazoneSPAm cross linkages-link- erswere containing also designed hydrazide and prepared groups (PHHZA)by researchers or amine [65–68] groups. Moreover, (PHEDA) two werePASP synthesizedAm cross-link- by Cha’sers containing team. PHHZA hydrazide and groupsPHEDA (PHHZA) can react or with amine the groupsaldehyde (PHEDA) groups wereof oxidized synthesized alginate by formingCha’s team. alginate PHHZA-based and hydrogels PHEDA via can hydrazone react with and the imine aldehyde linkages, groups respectively of oxidized [69] alginate. In addition,forming Kataoka’s alginate- teambased synthesized hydrogels via three hydrazone pH-responsive and imine charge linkages,-conversional respectively PASP [69]Am. deriv-In ad- ativesdition, containing Kataoka’s teamcis-aconitic synthesized amide three linkage pH (-PAsp(DETresponsive- Aco)charge [70]-conversional, PEG-PAsp(DET PASP-AmAco) deriv- [71]) oratives 2-propionic containing-3- methylcis-aconitic maleic amide amide linkage linkage (PAsp(DET (PEG-PAsp(DET-Aco) [70],- PMM)PEG-PAsp(DET [71]). The-Aco) obtained [71]) threeor 2-propionic polymers- 3showed-methyl negative maleic amide charge linkage at pH 7.4 (PEG due-PAsp(DET to the carboxylic-PMM) group [71]). inThe cis -obtainedaconityl (Aco)three polymersor 2-propionic showed-3-methyl negative maleic charge (PMM) at pH moieties, 7.4 due to bu thet exhibited carboxylic positive group incharge cis-aconityl at pH 5.5(Aco) because or 2-propionic they can return-3-methyl to PAsp(DET) maleic (PMM) or PEG moieties,-PAsp(DET) but exhibited via the breakup positive ofcharge cis-aconitic at pH amide5.5 because linkage they or can 2-propionic return to -PAsp(DET)3-methyl maleic or PEG amide-PAsp(DET) linkage viaunder the breakupacidic conditions. of cis-aconitic The amide linkage or 2-propionic-3-methyl maleic amide linkage under acidic conditions. The

Int. J. Mol. Sci. 2021, 22, 8817 6 of 18

preparation of pH-responsive polymers. The most well-known pH-responsive PASPAm derivative is NC-6300, in which EPI is bound to a PEG-PASPAm block copolymer through hydrazone linkage [23]. Thus, EPI can be released under acidic environments, such as in lysosomes. Doxorubicin (DOX)-conjugated PASPAm derivatives with hydrazone linkages were also designed and prepared by researchers [65–68]. Moreover, two PASPAm cross- linkers containing hydrazide groups (PHHZA) or amine groups (PHEDA) were synthesized by Cha’s team. PHHZA and PHEDA can react with the aldehyde groups of oxidized alginate forming alginate-based hydrogels via hydrazone and imine linkages, respectively [69]. In addition, Kataoka’s team synthesized three pH-responsive charge-conversional PASPAm derivatives containing cis-aconitic amide linkage (PAsp(DET-Aco) [70], PEG-PAsp(DET- Aco) [71]) or 2-propionic-3-methyl maleic amide linkage (PEG-PAsp(DET-PMM) [71]). The obtained three polymers showed negative charge at pH 7.4 due to the carboxylic group in cis-aconityl (Aco) or 2-propionic-3-methyl maleic (PMM) moieties, but exhibited positive charge at pH 5.5 because they can return to PAsp(DET) or PEG-PAsp(DET) via the breakup of cis-aconitic amide linkage or 2-propionic-3-methyl maleic amide linkage under acidic conditions. The pH-cleavable linkages used for preparing pH responsive PASPAm derivatives are summarized in Table2. Int. J. Mol. Sci. 2021, 22, 8817 7 of 18

Table 2. A summary of pH-responsive PASPAm derivatives containing pH-cleavable linkages.

pH Responsive Formation of Linkages between Linkages Ref PASPAm Derivatives AB Hydrazide group of PEG polyaspartate block NC-6300 Hydrazone Ketone group of EPI [23] copolymer Cross-linked nanocapsules with PADH and PACA Hydrazone Hydrazide group of PADH Aldehyde groups of PACA [56] PALHy-hyd-DOX Hydrazone Hydrazide group of PALHy Ketone group of DOX [65] ALN-PEG/C-18/HYD-DOX-g-PASPAM Hydrazone Hydrazide group of PASPAM Ketone group of DOX [66] Hydrazide group of MPEG/CA10/DOX-g-PASPAM Hydrazone Ketone group of DOX [67] MPEG/Hyd/CA10-g-PASPAM Hydrazide group of PA Hydrazone Ketone group of DOX [68] biotin-PEG/C18-PSI/Hydrazine PEG/Hyd-Curcumin/C18-g-PSI (NFA-Cur) Hydrazone Hydrazide group of PEG/C18-g-PSI Ketone group of Curcumin [60] Injectable PAsp hydrogel Hydrazone Hydrazide group of PAHy Aldehyde groups of PAAld [61] PHHZA-linked alginate hydrogels Hydrazone Hydrazide group of PHHZA Aldehyde groups of oxidized alginate [69] PHEDA-linked alginate hydrogels Imine Amino group of PHEDA Aldehyde groups of oxidized alginate [69] Aldehyde group of 4-adamantane carboxylate Pasp-EDA-g-Ad/mPEG Imine Amino group of Pasp-EDA [63] benzaldehyde PAsp(DET-Aco) cis-Aconitic amide Amino group of PAsp(DET) Anhydride group of cis-aconitic anhydride [70] PEG-PAsp(DET-ACO) cis-Aconitic amide Amino group of PEG-PAsp(DET) Anhydride group of cis-aconitic anhydride [71] 2-propionic-3-methyl Anhydride group of 2-propionic-3-methyl maleic PEG-PAsp(DET-PMM) Amino group of PEG-PAsp(DET) [71] maleic amide anhydride Anhydride group of hexahydrophthalic mPEG-g-P(ae-Asp)-Hap β-carboxylic amide Amino group of PEG-g-P(ae-Asp) [72] anhydride Int.Int. J.J. Mol.Mol. Sci.Sci. 2021,, 22,, 8817x FOR PEER REVIEW 88 of 1818

Besides the two above mentioned strategies, the unreacted succinimide rings in PAS- Besides the two above mentioned strategies, the unreacted succinimide rings in PAS- PAm derivatives also can be utilized to design pH-responsive controlled-release delivery PAm derivatives also can be utilized to design pH-responsive controlled-release delivery vehicles, because the hydrophobic succinimide units can be hydrolyzed at pH above 7.0 to vehicles, because the hydrophobic succinimide units can be hydrolyzed at pH above 7.0 form hydrophilic aspartic acid units, thereby releasing the encapsulated materials due to to form hydrophilic aspartic acid units, thereby releasing the encapsulated materials due the hydrophobic-to-hydrophilic change of PASPAm derivatives [73]. to the hydrophobic-to-hydrophilic change of PASPAm derivatives [73]. Interestingly, it has been reported that some temperature-responsive PASPAm deriva- Interestingly, it has been reported that some temperature-responsive PASPAm deriv- tives also exhibit pH responsiveness even without introducing pH-responsive functional groupsatives also or pH-cleavableexhibit pH responsiveness linkages. The even pH responsiveness without introducing of temperature-responsive pH-responsive functional PAS- PAmgroups derivatives or pH-cleavable may belinkage relateds. The to thepH presenceresponsiveness of hydroxyl of temperature groups in-responsive alkanolamide PAS- pendantsPAm derivatives [36] or themay end be groupsrelated (carboxylto the presence and amine of hydroxyl groups) groups of PASPAm in alkanolamide backbone [43pen-]. dants [36] or the end groups (carboxyl and amine groups) of PASPAm backbone [43]. 2.3. Redox-Responsive Poly(aspartamide) Derivatives 2.3. RedoxPASPAm-Responsive derivatives Poly(aspartamide) generally can Derivatives be endowed with redox-responsiveness by intro- ducingPA disulfideSPAm derivatives linkage generally in their backbone can be endowed (Figure with2a) orredox side-responsiveness chains (Figure by2b). introduc- Chu eting al. disulfide [47] used linkage cystamine-modified in their backbone mPEG (Figure as a macroinitiator2a) or side chains to initiate(Figure the 2b). polymerization Chu et al. [47] ofused BLA-NCA, cystamine and-modified then obtainedmPEG as a a redox-responsive macroinitiator to initiate mPEG-block-PASPAm the polymerization derivative, of BLA- whichNCA, and contains then aobtained disulfide a linkageredox-responsive between mPEG mPEG segment-block-PA andSPAm PASPAm derivative, segment. which Redox- con- responsivetains a disulfide PASPAm linkage derivatives, between mPEG which segment have mPEG and PASPAm pendants segment. with disulfide Redox-responsive linkages, canPASPAm also be derivatives, synthesized which through have grafting mPEG pendan 3,3’-dithiodipropionicts with disulfide acid-modified linkages, can also mPEG be syn- [74] orthesized the aminolysis through grafting reaction 3,3 between’-dithiodipropionic PSI and amino-terminated acid-modified mPEG disulfide [74] or functionalized the aminolysis mPEGreaction [ 48between]. Moreover, PSI and some amino cationic-terminated polymers disulfide [75,76 functionalized] or other compounds mPEG [48] [77. Moreover,] can also besome functionalized cationic polymers with [75,76] cystamine or other and compounds then used to [77] design can also reduction-responsive be functionalized with PASPAm cysta- derivativesmine and then for used DNA/RNA to design delivery. reduction Of- course,responsive cystamine PASPAm also derivatives can be used for as DNA/RNA a cross-linker de- forlivery. preparing Of course, reduction-responsive cystamine also can be nanogels used as a [cross78] or-linker nanoparticles for preparing [33 reduction] (Figure2-respon-c). In additionsive nanogels to S-S [78] linkage, or nanoparticles Se-Se linkage [33] is (Figure also a good 2c). In strategy addition for to designing S-S linkage, redox-responsive Se-Se linkage is

PASPAmalso a good derivatives strategy for [79 designing]. redox-responsive PASPAm derivatives [79].

S S

S - S S

Figure 2. The redox-responsiveredox-responsive P PASPAmASPAm derivativesderivatives containing disulﬁdedisulfide linkages ( a) in backbone and (b)) inin sideside chains,chains, andand disulﬁdedisulfide cross-linkedcross-linked ((cc)) nanogelsnanogels oror nanoparticlesnanoparticles andand ((dd)) hydrogels.hydrogels.

In addition, thiol thiol-disulfide-disulfide inter inter-conversion-conversion can can also also be be used used for for preparing preparing redox redox--re- responsivesponsive PASPAm PASPAm hydrogel hydrogelss [80– [83]80– or83 stabilized] or stabilized disulfide disulfide cross-linked cross-linked nanoparticles nanoparti- [84]. clesFor instance, [84]. For instance,PSI first reacted PSI first with reacted cysteamine, with cysteamine, and then and further then furtherreacted reactedwith other with amine other aminecompounds compounds (e.g., N (e.g.,,N-dimethylN,N-dimethyl-2-aminoethyl-2-aminoethyl [85], 6- [monodeoxy85], 6-monodeoxy-6-monoamino--6-monoamino-beta-cyclodextrin hydrochloride [86]) to obtain thiolated PASPAm derivatives. The obtained

Int. J. Mol. Sci. 2021, 22, 8817 9 of 18

beta-cyclodextrin hydrochloride [86]) to obtain thiolated PASPAm derivatives. The obtained thiolated PASPAm derivatives can be cross-linked by the oxidation of the thiol groups in their cysteamide pendants and formed reduction-responsive hydrogels (Figure2d).

2.4. Other Stimuli-Responsive Poly(aspartamide) Derivatives Besides the above three stimuli-responsive (temperature, pH and redox) PASPAm derivatives, PASPAm derivatives with other stimuli responsiveness (including light responsiveness and carbon dioxide (CO2) responsiveness) have also been investigated in the past two decades. CO2 is an inexpensive gas and also an important metabolite in cells. It is reported that polymers containing dialkylamine [87,88], amidine [89] or guanidine [90] moieties can exhibit reversible CO2-responsive behavior because CO2 can be bonded onto these polymers in the presence of water. For example, Kim’s group prepared three CO2-responsive PASPAm derivatives, PHEA-HIS [54], PHEA-Larg and PolyAspAm(OA/Larg) [91]. The swelling degree of cross-linked PHEA-HIS hydrogel decreased to about 18 when CO2 was bubbled into the solution containing PHEA-HIS hydrogel samples, and returned to about 50 after bubbling N2 gas. PHEA-Larg hydrogels and PolyAspAm(OA/Larg) nanoparticle also exhibited similar reversible volume change behavior by CO2/N2 purge. Because light is a clean stimulus and can be easily triggered on or off, light-responsive polymers have shown huge potential for a variety of applications. For designing light- responsive polymers, photo-cleavage groups such as o-nitrobenzyl [92] were generally introduced. For instance, o-nitrobenzyl (NB) alcohol [35] was ﬁrst activated by N,N0- carbonyldiimidazole and then grafted onto the pendant pentanolamide moieties of PHPA. When the obtained light-responsive NB-g-PHPA was treated under UV light at 365 nm for 30 min, the photo-cleavage o-nitrobenzyl carbonate ester linkage was broken and byproduct o-nitrosobenzaldehyde was detected. In addition, light can also be used for cross-linking to stabilize PASPAm-based micelles. For instance, light responsive coumarin moieties were introduced to the side chains of PASPAm derivative, and the formed micelles can be conveniently cross-linked and stabilized by exposing it to the light of 365 nm for 30 min [93]. Photo-crosslinking can also be achieved by introducing methacryloyl [9,94] or acryloyl [95] moieties to the ethanolamide or hydrazide pendants of PASPAm. Dual or multi-responsive PASPAm derivatives can also be prepared by the combina- tion of above-mentioned strategies for endowing various stimuli-responsiveness. Therefore, we will not go into the details here.

3. Applications Due to their good biocompatibility and biodegradability, PASPAm derivatives could be regarded as potential and promising materials and can be utilized in various areas of biomedical applications, such as drug carriers. As a drug carrier, the process of drug loading and drug release are both important. PASPAm derivatives with stimuli-responsiveness can usually show convenient drug loading methods or excellent controlled drug release per- formances. Therefore, this part focuses on the applications of stimuli-responsive PASPAm derivatives as drug carriers from two aspects: drug loading and drug release.

3.1. Drug Loading Amphiphilic polymers, which can self-assemble to form nanoparticles including micelles and vesicles, are one of the most important classes of polymers used to deliver hydrophobic drugs such as paclitaxel (PTX) and doxorubicin (DOX). For preparing drug- loaded nanoparticles, one of the important methods is dialysis. However, the dialysis method often takes a long time (at least a few hours) and requires the use of toxic organic solvents (e.g., DMSO, CH3OH) to dissolve the polymeric carrier [93], and the drug loading efﬁciency is usually low. Thus, a simple, easy-to-implement drug loading strategy with high efﬁciency is needed for preparing drug-loaded nanoparticles. Int. J. Mol. Sci. 2021, 22, 8817 10 of 18 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 18

Temperature and pH are often used as triggers for drug loading. For temperature Temperatureresponsive and pH polymers, are often theused general as triggers drug for loading drug processloading. isFor as temperature follows: (1) preparere- polymer sponsive polymers,aqueous the general solution drug of aloading certain process concentration is as follows: in PBS (1) prepare at a temperature polymer aque- (e.g., 4 ◦C) lower ous solution ofthan a certain LCST; concentration (2) add high-concentration in PBS at a temperature drug solution (e.g., 4 ° (e.g.,C) lower 20 mg/mL) than LCST; to the prepared (2) add high-concentrationtemperature responsivedrug solution polymer (e.g., 20 aqueous mg/mL) solution; to the prepared (3) heat temperature the mixture ofre- drug solution sponsive polymerand polymer aqueous aqueous solution; solution (3) heatimmediately the mixture with of drug about solution 1 min incubation and polymer by soaking into aqueous solutiona water immediately bath or with other about ways 1 (temperaturemin incubation higher by soak thaning LCST,into a water such asbath 60 or◦C); (4) slowly other ways (temperaturecool down higher the mixture than LCST, to room such temperature as 60 °C); (4)and slowly then cool filter down the the mixture mixture to remove non- to room temperatureentrapped and hydrophobic then filter the drug. mixture The process to remove of preparing non-entrapped drug-loaded hydrophobic nanoparticles from drug. The processthe temperature-responsiveof preparing drug-loaded polymer nanoparticles aqueous from solution the temperature is called the-responsive quick heating method. polymer aqueousFor example,solution is Jiang called et the al. reportedquick heating that PTXmethod. was successfullyFor example, loaded Jiang et into al. re- micelles formed ported that PTXby was temperature-responsive successfully loaded PASPAminto micelles derivatives formed throughby temperature quick heating-responsive method, avoiding PASPAm derivativesthe use through of toxic quick organic heating solvents method [35,96, avoiding,97]. Using the use temperature-responsive of toxic organic sol- PEGylation vents [35,96,97]phe-g-PHPA. Using temperature as drug-responsive carrier, the PEGylation PTX-loading phe capacity-g-PHPA was as drug high, carrier, up to 29%,the with a high PTX-loading capacityloading was efficiency high, up of to 99% 29% [97, with]. Using a high pH-responsive loading efficiency PASPAm of 99% derivatives, [97]. Using the drug also pH-responsivecan PASPAm be loaded derivatives, into micelles the drug by quickly also can changing be loaded the into pH ofmicelles the polymer by quickly aqueous solution changing the pHfrom of belowthe polymer to above aqueous pHt. Forsolution instance, from the below aqueous to above mixture pHt. containingFor instance, pH-responsive the aqueous mixturephe/DEAE-g-PHPA-g-mPEG containing pH-responsive and phe/DEAE doxorubicin-g-PHPA hydrochloride-g-mPEG wasand prepareddoxoru- at pH value bicin hydrochloridebelow was pH tpreparedfirst (pH at = pH 4.0), value and below then apH certaint first (pH volume = 4.0), of and weakly then alkalinea certain PBS solution volume of weakly(pH =alkaline 8.2) was PBS added solution to adjust (pH = the 8.2) pH was above added pH tto. After adjust filtering the pH to above remove pH non-entrappedt. After filtering DOX,to remove DOX-loaded non-entrapped micelles DOX, prepared DOX- withloaded pH-responsive micelles prepared phe/DEAE-g-PHPA-g-mPEG with pH-responsive phe/DEAEwere obtained-g-PHPA [34-g-].mPEG Therefore, were temperature-responsiveobtained [34]. Therefore, and temperature pH-responsive-respon- PASPAm deriva- sive and pH-responsivetives play anPASPAm important derivatives role in the play drug an loadingimportant process role in because the drug they loading can achieve a rapid process becausedrug they loading can achieve procedure a rapid (Figure drug3 ).loading procedure (Figure 3).

FigureFigure 3. 3. TypicalTypical drug drug loading loading procedures procedures of of various various stimuli stimuli-responsive-responsive PASPAm PASPAm derivatives derivatives..

Hydrogels areHydrogels also an important are also anclass important of polymers class that of polymerscan be used that as can drug be carriers. used as drug carriers. Cross-linking Cross-linkingis a well-known is astrategy well-known for preparing strategy fordrug preparing-loaded hydrogels drug-loaded or nanogels. hydrogels or nanogels. The cross-linkingThe reac cross-linkingtion can occur reaction between can occur two polymers between two containing polymers reactive containing functional reactive functional groups [13,69,81]groups, or between [13,69,81 polymer], or between and small polymer molecule and small cross molecule-linker [98 cross-linker–101]. Of course, [98–101 ]. Of course, enzymes can also be used as cross-linker to promote the cross-linking reaction of PASPAm

Int. J. Mol. Sci. 2021, 22, 8817 11 of 18

enzymes can also be used as cross-linker to promote the cross-linking reaction of PASPAm derivatives for the preparation of nanogels [102]. For PASPAm derivatives without stimuli- responsiveness, another option is the precipitation of the polymer in physiological medium to form gel-like depot. The drug and polymer can be dissolved in an organic solvent with high miscibility with water, and then the resulting mixture solution was slowly injected into physiological medium to allow the formation of drug-loaded hydrogels. According to this strategy, Fiorica et al. successfully prepared a sulpiride-loaded gel-like depot using PASPAm-polylactide copolymer, in which N-methyl-2-pyrrolidone was employed to dissolve sulpiride (an antipsychotic drug) and PASPAm-polylactide copolymer due to its low systemic toxicity [14]. This sulpiride-loaded depot can prolong a sustained release of sulpiride in vitro for about one week. For PASPAm derivatives that respond to stimuli, even if organic solvents are not used, drug-loaded hydrogels can also be prepared, which will reduce the potential risks caused by the use of organic solvents. For instance, Cao et al. successfully prepared ketoprofen-loaded hydrogels by irradiation of the mixture of light-responsive PASP-Hy-AC aqueous solution and ketoprofen (a nonsteroidal anti-inflammatory drug) under long-wavelength UV for 1 h, and the obtained ketoprofen-loaded hydrogel exhibited the sustained ketoprofen release for about 50 h [95]. Redox-responsive PASPAm derivatives containing thiol groups can also be used for drug-loading. For example, PASP-CEA was dissolved in the PBS containing ofloxacin (a second-generation fluoroquinolone antibiotic), and then oxidant solution was added to ofloxacin/PASP-CEA PBS solution to form ofloxacin-loaded hydrogel via the oxidation of the thiol groups [83]. Moreover, some temperature-responsive polymer aqueous solutions can turn into a gel state in a short time (a few minutes) when heated. Thus, the formation of drug-loaded hydrogels based on temperature-responsive polymers, such as chitosan/β-glycerophosphate [103,104] and Pluronic F127 [105], can be triggered by the temperature difference between the external environment and the body. However, there is no report on the use of temperature-responsive PASPAm derivatives for preparing drug loaded hydrogels, although there are many reported temperature-responsive PASPAm derivatives. Polymer-drug conjugates composed of drug molecules covalently linked to polymeric carriers are also an important part of drug delivery systems. Because the drug and the polymer carrier are covalently linked, polymer-drug conjugates generally do not require response to external stimuli to achieve drug loading.

3.2. Drug Release As drug carriers, it is also very important that they can release drugs to the desired locations in response to speciﬁc stimuli. Thus, stimuli-responsive polymers also play a key role in the drug release process. For drug release, pH and redox are the most investigated triggers due to the acidic environment around the tumor (pH range of 5.5–7.0) [106] and in the endosome (pH ≈ 5.0) [107] and the presence of reduced glutathione (GSH) [108]. Although polymer-drug conjugates are usually not required to respond to external stimuli during drug loading, they are often required to respond to external stimuli during the drug release process to deliver the drug to the desired location. NC-6300, which has already undergone Phase I (19 subjects) [109] and Phase 1b (29 subjects) [110] clinical trials, is the most famous stimuli-responsive polymer-drug conjugate based on PASPAm. The EPI conjugated with hydrazone linkage in NC-6300 can be released under acidic environment around tumors, so it exhibits selective tumor accumulation. Clinical trial results showed that NC-6300 was well tolerated in patients with various solid tumors including urothelial carcinoma, breast cancer, cholangiocarcinoma and leiomyosarcom, and its toxicity was lower than conventional EPI formulations. The maximum tolerated dose and recommended phase 2 dose of NC-6300 obtained in Phase 1b were 185 mg/m2 and 150 mg/m2, respectively, which are higher than conventional EPI dose. Besides EPI, DOX was another widely investigated anti-tumor drug in polymer-drug conjugates as a drug delivery system. For instance, Lim et al. conjugated DOX to alendronate (ALN)-modiﬁed Int. J. Mol. Sci. 2021, 22, 8817 12 of 18

PASPAm derivatives with hydrazone linkages to obtain polymer-DOX conjugate ALN- PEG/C18/HYD-DOX-g-PASPAM, and prepared bone targeting nanoparticles. Results showed that 75% of the conjugated DOX was released from the prepared polymer-DOX conjugate at pH 5.0 due to the cleavage of hydrazone bonds, and the volume of tumor decreased significantly to 1550 mm3 after treatment with ALN-PEG/C18/HYD-DOX-g- PASPAM (control sample in PBS, 3850 mm3)[66]. The pH-controlled release of DOX in other PASPAm-DOX conjugates containing hydrazone linkages were also studied, results showed that the amount of DOX released at pH 5.0 was significantly more than that at pH 7.4 in the same time period [65,67,68]. Chang et al. conjugated PTX to the end group (-NH2) of the backbone of PEG-b-PBLA via a reduction-responsive disulfide linkage using 3,30-dithiodipropionic acid as a coupling agent, and results showed that the release rate of PTX in an environment with 10 mM GSH (mimicking the environment in tumor cells) was significantly higher than that in an environment with 2 µM GSH (mimicking the environment in human blood) [111]. Therefore, the introduction of stimuli-responsive cleavable linkages is a key strategy for the controlled drug release of polymer-drug conjugates. For nanoparticle drug delivery systems prepared from amphiphilic polymers, the response to environmental stimuli generally can break the hydrophobicity-hydrophilicity balance, thereby disturbing the stability of the drug-loaded nanoparticles to achieve the aim of drug release. Gu et al. prepared DOX-loaded nanoparticles by dialysis using temperature-responsive PAIPAHA as polymeric carrier, and their DOX release profiles evaluated at different temperature showed that the release amount and rate of DOX at 37 ◦C (human body temperature, 45% within 10 h) were both higher than that at 25 ◦C (room temperature, 35% after 110 h) [27]. This may be caused by the LCST of PAIPAHA (30 ◦C) being between room temperature and body temperature. When the environmental temperature is higher than the LCST of PAIPAHA, the formed nanoparticles were disturbed and became unstable due to the weakened hydrogen-bonding or hydrophobic interactions between PAIPAHA and DOX. Thus, temperature can be used as a switch to control the drug release. The response to pH or GSH can also be used to realize the controlled release of drug from nanoparticles. Cai et al. prepared a disulfide cross-linked micellar nanodrug loaded with sorafenib from pH/reduction dual-responsive mPEG-PAsp (MEA&DIP), and realized controlled release of sorafenib using pH and GSH as the switch [112]. Due to the disulfide cross-linking, this sorafenib-loaded nanodrug can remain stable in blood circulation, but it can also rapidly release sorafenib inside cancer cells due to GSH-induced disulfide bond breakage and the protonation of DIP (2-aminoethyldiisopropylamide) pendants in an acidic environment (leading to a hydrophobic-to-hydrophilic change). Light with specific wavelength also can be used as a stimulus for controlled drug release. After 15 min of irradiation at 365 nm, PTX-loaded nanoparticles containing photo-cleavable o-nitrobenzyl carbonate ester linkages showed an significantly faster PTX release behavior [35]. For hydrogels, drug release generally can be controlled by the degree of swelling, and redox is one of the most investigated triggers due to the reductive environment in tumor tissue (10 mM GSH). Krisch et al. chose fluorescein isothiocyanate-dextran as model drug to investigate the drug release behavior. Results reveal that a reductive environment can promote the release of model drug from S-S cross-linked PASPAm hydrogel, because the degree of swelling increased (10 mM DTT in PBS) due to the cleavage of S-S linkages [81]. Park et al. prepared DOX-loaded PASPAm nanogels, the release rate of DOX in reductive environment was significantly faster than that in non-reductive environment due to the disintegration of nanogels [78].

4. Degradation The degradability of PASPAm derivatives is based on the hydrolysis process of amide linkages in their backbone. Many studies have conﬁrmed that the backbone of PASPAm derivatives can be degraded by bacteria (i.e., strain KP-2: JCM10638, 15 days) [113,114], enzyme (i.e., hydrolase puriﬁed from Sphingomonas sp. KT-1, β-amide linkage) [115,116] or activated sludge (28 days) [117,118] after a few days. For PASPAm derivatives that Int. J. Mol. Sci. 2021, 22, 8817 13 of 18

respond to stimuli, the degradation site and behavior are more diverse. For instance, Juriga et al. [119,120] investigated the degradability of cross-linked PASPAm hydrogels containing disulﬁde linkages with different enzymes (trypsin-EDTA, dispase and collagenase I). Collagenase I showed the best effect for the degradation of hydrogel disk, followed by trypsin-EDTA and then dispase, because collagenase I cleaved the disulﬁde linkages in the hydrogel. Interestingly, the degradation of PASPAm derivatives containing pH- responsive amino groups can be achieved without enzyme due to the nucleophilic attack of the N in CONHside at the C in CONHmain [121], and can be affected by their pendant moieties. Naito et al. [7] reported that the bearing pendants with a longer alkyl spacer (i.e., 3-aminopropylamide (3 carbons), 4-aminobutylamide (4 carbons)) showed more strongly suppressed degradation than that with a shorter alky spacer (i.e., 2-aminoethylamide (2 carbons)). Although the degradation of PASPAm derivatives has been widely investigated, so far there have been no in vivo studies on their degradation.

5. Conclusions With the ever-increasing demand for novel smart materials in biomedical fields, the development of stimuli-responsive PASPAm derivatives has attracted more and more attention due to their biocompatibility and biodegradability. From a synthesis point of view, most stimuli-responsive PASPAm derivatives can be easily prepared by introducing functional groups or moieties that respond to various stimuli, but it is worth not- ing that temperature-responsive PASPAm derivatives can also be synthesized without introducing specific temperature-responsive moieties. In addition, some temperature- responsive PASPAm derivatives possess pH responsiveness even without introducing pH-responsive functional groups or pH-cleavable linkages. Thus, the pH response mech- anism of temperature-responsive PASPAm derivatives needs to be studied in depth in the future. From an application point of view, stimuli-responsive PASPAm-based drug delivery systems including nanoparticles, hydrogels and polymer-drug conjugates are reviewed in this paper from two aspects: drug loading and controlled release. External stimuli can not only be utilized to achieve convenient drug loading but also controlled drug release. Temperature and pH generally can be used as triggers to realize drug loading for nanoparticles, while thiol groups and light with specific wavelength usually can be used as triggers to prepare drug-loaded hydrogels. Although temperature has been reported to trigger the preparation of drug-loaded hydrogels by other temperature-responsive materials, there is no report on the use of temperature-responsive PASPAm derivatives to prepare drug-loaded hydrogels. Considering the advantages of the stimuli-triggered drug loading method (avoiding the use of toxic organic solvents and high drug loading efficiency), such studies may be an interesting area in drug delivery systems. For controlled drug release, it can be triggered by most of stimuli, such as temperature, pH, GSH and light. NC-6300, which exhibits selective release of EPI into tumor tissue, is a typical representative of stimuli-responsive polymer-drug conjugates for drug delivery based on PASPAm derivatives. In conclusion, stimuli-responsive PASPAm derivatives hold great prospects as drug carriers. Although some considerable progress has been made, there are still some issues that have not yet been clarified in terms of response mechanisms. Taking account of the growing interest in stimuli-responsive polymers, more interesting stimuli-responsive PASPAm derivatives may be designed and applied for drug delivery in the future.

Author Contributions: Conceptualization, G.Z.; writing—original draft preparation, G.Z., H.Y. and C.B.; writing—review and editing, G.Z., H.Y. and C.B. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Research Foundation of Hubei Provincial Key Laboratory of Green Materials for Light Industry and Collaborative Innovation Center of Green Light-weight Materials and Processing (201907B08). Int. J. Mol. Sci. 2021, 22, 8817 14 of 18

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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