Polymerization-Induced Self-Assembly for Efficient Fabrication of Biomedical Nanoplatforms

Amphiphilic copolymers can self-assemble into nano-objects in aqueous solution. However, the self-assembly process is usually performed in a diluted solution (<1 wt%), which greatly limits scale-up production and further biomedical applications. With recent development of controlled polymerization techniques, polymerization-induced self-assembly (PISA) has emerged as an efficient approach for facile fabrication of nano-sized structures with a high concentration as high as 50 wt%. In this review, after the introduction, various polymerization method-mediated PISAs that include nitroxide-mediated polymerization-mediated PISA (NMP-PISA), reversible addition-fragmentation chain transfer polymerization-mediated PISA (RAFT-PISA), atom transfer radical polymerization-mediated PISA (ATRP-PISA), and ring-opening polymerization-mediated PISA (ROP-PISA) are discussed carefully. Afterward, recent biomedical applications of PISA are illustrated from the following aspects, i.e., bioimaging, disease treatment, biocatalysis, and antimicrobial. In the end, current achievements and future perspectives of PISA are given. It is envisioned that PISA strategy can bring great chance for future design and construction of functional nano-vehicles.


Introduction
In recent years, nanoparticles (NPs) that can be precisely de signed and manipulated have been applied in a range of fields including catalysis, energy storage and conversion, and drug delivery [1][2][3][4][5][6][7]. Polymeric NPs are appealing materials for bio medical applications owing to their excellent biocompatibility and tailorability. Particularly, amphiphilic copolymers can simul taneously selfassemble into various nanostructures in aqueous solution [8]. Notably, Zhang and Eisenberg first reported the selfassembly behavior of amphiphilic block copolymers (BCP) in solution in 1995 [9]. Since then, various NPs including spheres, worms, and vesicles have been discovered [10][11][12]. However, the process is usually performed in dilute solution (<1 wt%). The efficiency and reproducibility of the preparation of polymeric NPs are unpleasant. These above adverse defects greatly restrict the largescale preparation of functional NPs and their applications. Therefore, it is quite demanding to find an alternative approach to prepare NPs efficiently.
Notably, polymerizationinduced selfassembly (PISA) that combines polymerization and selfassembly in one pot has shown many distinct advantages, such as laborsaving, repeat ability, and high solid concentration as high as 50 wt% [13][14][15][16][17]. By utilization of suitable solvent, soluble macroinitiator can be chainextended by polymerization with the second monomers. As the polymerization proceeds, the second insoluble block gradually drives the selfassembly of polymer chains. Armes and coworkers summarized the PISA process under the differ ent conditions [18,19]. The morphology of obtained NPs can be well regulated during the PISA process [20][21][22]. Notably, the morphology of selfassembled NPs can be finely tuned by adjusting the packing parameter (P). P can be given by the equation P = v/al, where a is the interfacial area of the hydro philic block and v and l are the volume and length of the hydro phobic block, respectively. When P ≤ 1/3, amphiphilic BCPs usually selfassemble into spherical micelles. Worms can be obtained when 1/3 < P ≤ 1/2, while vesicles are formed when 1/2 < P ≤ 1. Interestingly, when P > 1, highlyordered inverse structures (e.g., spongosome and cubosome) can be fabricated. Pan and Du reported that the PISA process has been succeeded by utilization of controlled polymerization tech niques includ ing nitroxidemediated radical polymerization (NMP), atom transfer radical polymerization (ATRP), reversible addition fragmentation chain transfer (RAFT) polymerization, and ring opening polymerization (ROP) [23,24].
Due to fine control over the morphology and size of the pre pared NPs, PISA has become a robust technique to fabricate NPs for various applications. As a new trend, growing functional groups have been gradually introduced into the PISA system to construct multifunctional nanoplatforms [25][26][27][28]. Notably, by incorporation of reactive groups, such as thiol group [29] and epoxy group [30], into hydrophilic chain or hydrophobic core, fabricated NPs can further conjugate with functional molecules. What is more, theranostic molecules, such as imaging agents, drugs, and proteins, can also be finely encapsulated during the selfassembly process, resulting in enhanced diagnostic and ther apeutic effects. In addition, enzymes with catalytic effect can also be combined with polymeric nanoreactors to achieve bio catalysis and antimicrobial action. Recently, these polymeric NPs manufactured by PISA have been emerged in biomedical field.
Recently, PISA has been reviewed by Armes, O'Reilly, Pan, Yuan, Couturaud, and Du, as well as other groups from various perspectives [19,23,24,[31][32][33][34]. Among these existing reviews, Pan and Du recently summarized the development of polym erization techniques in PISA [23,24]. Yuan and Armes focused on PISA by nonthermal initiation, which was beneficial to the loading of biomacromolecules [14,33]. Couturaud and Pan also reviewed the application of PISA for drug delivery [32,34]. However, none of them focused on the fabrication of nanoplat forms by PISA for biomedical applications. As an efficient tool to prepare functional NPs, fabricating biomedical nanoplat forms by PISA is worthy of much attention.
In this review, after the introduction, the principles and fea tures of various PISA systems are demonstrated with depth ( Fig. 1). Afterward, updated biomedical nanoplatforms built by PISA strategy are reviewed from 4 aspects, i.e., bioimaging, Fig. 1. Fabrication of nanoplatforms by polymerization-induced self-assembly and their biomedical applications. Reproduced with permission from [136]. Copyright 2018, Royal Society of Chemistry. Reproduced with permission from [143]. Copyright 2022, Royal Society of Chemistry. Reproduced with permission from [159]. Copyright 2018, American Chemical Society. Reproduced with permission from [164]. Copyright 2017, American Chemical Society. Reproduced with permission from [169]. Copyright 2020, American Chemical Society. Reproduced with permission from [170]. Copyright 2019, Royal Society of Chemistry. disease treatment, biocatalysis, and antimicrobial. In the end, some conclusions and perspectives for further development of PISA are elaborated carefully. It is believed that more and more fascinating polymeric nanoplatforms for other applications can be constructed by PISA in the future.

Preparation and Characterization of Nanoplatforms by PISA NMP-PISA
NMP is one of the earliest discovered reversibledeactivation radical polymerization (RDRP) techniques, and the reaction process can achieve controlled/active polymerization by the utilization of 2,2,6,6tetramethylpiperidine (TEMPO) or its de rivatives (Fig. 2). The first work about NMPPISA was reported in 2005 by Charleux's group [35]. Briefly, styrene (St) and nbutyl acrylate (BA) monomers were grafted from the end of Ntert butylN(1diethyl phosphono2,2dimethyl propyl) nitroxide (SG1)terminated poly(sodium acrylate) in water at 120 °C under 3 bar pressure of nitrogen. With the growth of hydrophobic chain, obtained amphiphilic BCPs selfassembled into spherical NPs in situ. The hydrodynamic diameter of polystyrene (PS) and poly(nbutyl acrylate) (PBA) particles was 65 and 90 nm, respec tively. Notably, when pH was varied from 7 to 4, the diameters of PS NPs decreased to 55 nm and the diameters of PBA NPs decreased to 76 nm. Notably, the variation of the diameters was due to the collapse of hydrophilic corona after protonation. This pioneering work laid the foundation for future research.
Later in 2009, the same group first reported pHsensitive vesicles, which were fabricated with high concentrations by NMP PISA under emulsion condition [36]. Poly(sodium acrylate) macroalkoxyamine (PNaASG1) was used to polymerize with 4vinylpyridine (4VP) monomers at pH 11, 120 °C under 3 bar pressure. Most monomers (>90%) were consumed within 4 h, as the polymerization proceeded, and spheres, wormlike mi celles, and spherical vesicles were observed. When the pH decreased from 11 to 2, the milky solution became clear, ascribed to the protonation of poly(4vinylpyridine) (P4VP) block. Therefore, these pHsensitive vesicles could efficiently release encapsulated drugs once upon acid environment. However, the disadvantages of NMP also existed in the above PISA process including high reaction temperature, which was not beneficial to the encapsu lation of biomolecules.
To simplify the NMPPISA process, some pioneering works have been reported. For this purpose, watersoluble brushtype macroalkoxyamine initiator poly (poly (ethylene oxide) methyl ether methacrylate-co-styrene) was used to initiate emulsion polymerization of nbutyl methacrylate and St units at 85 °C [37]. The effect of the macroinitiator concentration, pH, and the salt concentration on the stability and morphology of selfassembled nanoobjects were studied. If salt concentration was at 0 or 1 mM, spherical NPs tended to be obtained. When salt concentration was increased to 10 mM or even 100 mM, elongated micelles and vesicles appeared. It was explained that the polymer became more hydrophilic with increased ionic strength, which could induce the transformation of the mor phology. Yoshida fabricated giant vesicles by NMPPISA under room temperature (RT) and ultraviolet (UV) irradiation instead of heating [38,39].
In addition to the study of polymeric assemblies, NMPPISA can also be used for the fabrication of polymer-inorganic hybrid systems [40]. For this purpose, macroalkoxyamine initiator was adsorbed on the surface of silica NPs to copolymerize with nbutyl methacrylate (BMA) and St units by NMP. As the hydro phobic block grew, amphiphilic BCPs selfassembled into various morphologies. Impressively, both the pH value and the size of silica NPs had important impact on the morphology of nanocomposites. Notably, by taking advantage of the cryotrans mission electron microscope (TEM) tech nique, the intermediate morphologies during the PISA process were captured, which was beneficial to understand the mechanism of the selfassembly behaviors.
Above all, although NMPPISA has been explored for many years, the literatures that have been reported remain sparse as a result of stringent experimental conditions and uncommon agents. Meanwhile, the reported monomers for NMPPISA are quite limited and possess poor biocompati bility and biodegradability.

RAFT-PISA
Among the controllable radical polymerization techniques, RAFT polymerization has been considered as a robust method to prepare welldefined polymers with desired structure and molecular weight (MW). Generally, the polymerization pro cess usually requires the usage of chain transfer agents (CTAs), such as dithioester derivatives [RC(=S)Z]. In recent years, much effort has been used to develop RAFTPISA [41,42]. In general, solvophilic macroCTAs should be first synthesized as stabilizer block and chainextended with the solvophobic block. With the increase of the solvophobic chain, amphiphilic BCPs selfassemble into NPs in situ.

Thermal-initiated RAFT-PISA
Among all these RAFTPISA systems, thermal initiation is the most wellestablished initiation method. Traditional initi ators [e.g., 2,2'azobisisobutyronitrile ( AIBN) or 4,4'azobis (4cyanovaleric acid) (ACVA)] can produce free radicals at high temperature (60 to 90 °C), which will initiate the polym erization process. Also, various kinds of amphiphilic copolymer based NPs have been successfully fabricated by thermal initiated RAFTPISA in dispersion or emulsion condition [52,[55][56][57][58]. The fabrication of these NPs can be predicted and guided by phase diagram [59,60]. Nowadays, ,many works were devoted to control the mor phology of obtained NPs. In addition to the significant effects of solid content and chain length on the morphology of NPs, other factors such as monomer solubility, temperature, pH, and complementary hydrogen bonding interaction were also studied [61][62][63][64][65]. Very recently, the effect of host-guest complexation interaction on the morphology has attracted much attention. For example, Yuan and coworkers first achieved aqueous dis persion polymerization with waterimmiscible monomers by using host-guest chemistry [46]. The ratio of cyclodextrin/ styrene (CD/St) was regulated to obtain various morphologies. When the CD/St molar ratio was set as 1:1, the dumbbell struc ture was observed with the increased degree of polymerization (DP) of PS block. By using host-guest chemistry, the library of monomers for RAFTPISA is greatly expanded. More recently, the same group successfully fabricated uniform polyethylene glycol (PEG)-b-PSt nanoflowers with low dispersity by increas ing the polymerization rate [45]. This strategy could also be applied to other PISA formulations to fabricate monodisperse and hierarchical NPs. In addition, Armes and coworkers fabri cated PEGdecorated vesicles by utilization of host-guest inter action between azobenzenecapped PEG and CDmodified vesicles to regulate the kinetics of morphological transition [66]. It was found that these supramolecular vesicles had a faster response to temperature than that of pure CDmodified vesicles, which may have great potential in design and fabrication of lightcontrolled drug delivery systems.
Although the thermal initiation is commonly used, the rel atively high temperature involved in the reaction process limits the scope of RAFTPISA. High temperature hampers the intro duction of some biological materials (e.g., enzyme), which are labile to heat. Therefore, exploring more moderate reaction conditions and simpler steps is a trend of PISA development. Nonthermally initiated RAFTPISA formulations (e.g., photo, enzyme, and ultrasound wave initiation) seem to have great potential in bioapplications.

Photo-initiated RAFT-PISA
Photoinitiated RAFT polymerization has attracted much atten tion owing to its robust control of polymerization in time and space [67][68][69][70]. Compared with thermal initiation, photo initiation can allow the polymerization process to take place at low temper ature. According to the wavelength of light, photo initiation mainly includes UV and visible light initiation. Photoinitiated RAFT polymerization is mild because it is usually conducted at RT.
Notably, Chen and coworkers creatively introduced photo initiated RAFT polymerization into the PISA system [71]. The macroCTA P4VP was chainextended with St units in methanol upon UV light, resulting in polymeric micelles at ambient tem perature. However, the polymerization kinetics showed that the monomer conversion was relatively low in this photo initiated polymerization process when compared with thermalinitiated dispersion polymerization, which was attributed to low activity of St at RT. In addition, when the MW did not increase upon UV light, the reaction could achieve secondary polymerization by heating. This interesting phenomenon may be explained that some encapsulated photoinitiator like AIBN cannot be irradiated by UV light due to the limited penetration depth. In general, the photoinitiated RAFTPISA expands the scope of PISA formu lations and shows promise as a powerful strategy to scale up preparation of polymeric NPs under mild conditions. Despite the successful preparation of polymeric NPs by UVinitiation PISA, the photolysis of the RAFT agent and loss of polymerization control by UV light are still the main problem. Therefore, visible light initiation is gradually becoming the new trend of photoinitiation. During the last decade, some research groups have reported visible lightinitiated RAFTPISA [72][73][74][75][76][77][78][79][80]. For instance, Zhang and coworkers reported the preparation of worms and vesicles by RAFTmediated emulsion polymerization under ambient temperature (Fig. 3A) [60]. Spe cially, tertbutyl methacrylate (tBMA) and tertbutyl acrylate (tBA) were used as coreforming monomers. Compared with tBMA, tBA tended to induce the evolution of morphology, which may be caused by the lower glass transition temperature (T g ) and weaker hydro phobicity (Fig. 3B). Furthermore, to over come the quench effect of oxygen, glucose oxidase (GOx) and glucose were used, result ing in the success of RAFTPISA in open vessels (Fig. 3C).
Photoinitiated RAFTPISA is promising for the incorpora tion of bioactive materials into NPs because of its mild reaction condition. Some research work had successfully synthesized polymeric NPs with hydrophilic shell consisting of poly(amino acid)based materials or polysaccharides [81][82][83]. For example, Gianneschi and coworkers fabricated proapoptotic peptide brush polymeric NPs by incorporating the proapoptotic "KLA" pep tides, which could induce the apoptosis of cancer cells by dis rupting the mitochondrial membrane [83]. Notably, the higher graft density of the KLA peptide in the obtained polymeric NPs had better anticancer efficiency according to the comparative experiment. Similarly, polysarcosine and polysaccharide have also been used as the hydrophilic shell for photoPISA to improve biocompatibility and biodegradability [81,82].
Furthermore, photoinitiated RAFTPISA can also be used as a robust technique to fabricate Janus inorganic/polymeric hybrid NPs. Janus gold NPs/block copolymers (Au@BCPs) have attracted much attention owing to their distinct physic ochemical properties. However, the high reaction temperature of conventional thermalinitiated RAFTPISA usually leads to AuNP aggregation. Therefore, photoinitiated RAFT PISA can be used as an alternative method to prepare Janus Au@BCP without high temperature. For this purpose, the P4VPCTA endcapped with trithiocarbonate group was first decorated on the surface of AuNPs [84]. During the PISA process, by adjusting the ratio of monomer to CTA and reaction time, the size and morphology of Janus Au@BCPs could be precisely controlled.
Besides, continuous flow reactor has recently been designed to prepare polymeric NPs massively [85]. The morphology can be precisely tuned by adjusting the light intensity, solid content, and residence time. Notably, the continuous process can pro duce 60 g of polymeric NPs in 24 h, which has great advantages over the batch reactions. More recently, GOx and glucose were added into the reaction mixture to achieve photoPISA without fussy degassing [86]. Through this strategy, polymeric NPs can be prepared on a large scale for biomedical application without batchtobatch difference.
In general, compared with thermalinitiated RAFTPISA, photoinitiated RAFTPISA has more potential in constructing polymeric NPs when it involves the encapsulation of active molecules, which is attributed to the mild reaction of photo PSIA [87]. According to the published literature, photoPISA was preferred to build biological platforms.

Enzyme-initiated RAFT-PISA
Despite the fact that photoinitiated RAFT polymerization can be performed on a mild condition, some drawbacks still exist. For instance, the limited depth of light penetration can cause the incomplete monomer conversion. In addition, photosen sitive materials are not suitable for this reaction system.
To date, enzymeinitiated RAFT polymerization has been successfully conducted [88][89][90][91]. These works realized a mild and efficient PISA by utilization of the horseradish peroxidase/ hydrogen peroxide/acetylacetone (HRP/H 2 O 2 /ACAC) ternary initiating system. In addition, GOx was also exploited to pro duce free radicals in the presence of oxygen [92,93]. For exam ple, Tan and coworkers first introduced enzyme initiation into RAFTPISA [94]. In this case, mPEG1134cyano4(ethylth iocarbonothioylthio) pentanoic acid (mPEG 113 CEPA) was used as macroCTA for the polymerization of 2hydroxypropyl methacrylate (HPMA) units (Fig. 4A). By adjusting the solid content and DP of the HPMA block, various morphologies can be obtained, and detailed phase diagram was drawn (Fig.  4B). During the reaction process, free radicals were generated through the oxidation of ACAC by hydrogen peroxide (H 2 O 2 ) in the presence of HRP. To avoid the influence of oxygen, GOx was used as catalyst to transform oxygen into H 2 O 2 in the presence of glucose (Fig. 4C). In this way, complicated freezepump-thaw cycles were not necessary. Notably, the vesicles had the ability to load both biomacromolecules and inorganic mate rials. As characterized by TEM, the SiO 2 NPs and bovine serum albumin (BSA) could be loaded in the cavities of polymeric vesicles, and the BSA had little effect on the formation of vesic ular morphology.
As known, the epoxy group is unstable under high tem perature (e.g., 80 °C). Therefore, traditional thermalinitiated RAFTPISA is not suitable for preparing epoxyfunctionalized NPs. Impressively, epoxyfunctionalized polymeric NPs can be prepared by photoinitiated RAFTPISA [95]. Besides, enzymeinitiated seeded RAFTPISA can also be used to prepare epoxyfunctionalized triblock copolymer vesicles [89]. In this case, the prepared poly(glycerol monometh acrylate) 46 PHPMA 300 vesicles were used as macroCTA for the polymerization of glycidyl methacrylate (GlyMA) mon omers, and the surface of vesicles became rough by increasing the DP of PGlyMA block.
In a word, enzymeinitiated RAFTPISA is a mild strategy to fabricate polymeric nanoobjects on a large scale. Compared with photoinitiated PISA, it expands the scope of monomers, which can be involved in the mild PISA process. However, the current enzymeinitiated PISA experiments were carried out in aqueous solution. Therefore, exploring more types of enzymes suitable for PISA and improving the stability of enzyme in organic solvent are worthy of further study.

Ultrasound-initiated RAFT-PISA
Compared with above RAFTPISA, ultrasoundinitiated RAFT PISA does not need traditional initiators [96]. Free radicals for ultrasoundinitiated RAFTPISA are generated by highfrequency ultrasound. Different from photoinitiated RAFTPISA, the free radicals dispersed evenly throughout the reaction system, avoid ing the problem of uneven distribution of free radical concen tration. In 2018, the first ultrasoundinitiated RAFTPISA was successfully performed in water by using highfrequency ultrasound (490 kHz) with monomer conversion nearly 100% in 60 min [97]. The obtained poly(poly(ethylene glycol) methyl ether acrylate)poly(Nisopropylacrylamide-co-N,N′ methylenebis(acrylamide)) (PPEGAP(NIPAM-co-MBA)) nano gels had the lower critical solution temperature (LCST)type thermosensitive characteristic. Because the LCST of PNIPAM block was 32 °C, when temperature was increased from 25 to 45 °C, the size and polydispersity index (PDI) of polymeric NPs decreased, which was caused by the collapse of P(NIPAMco-MBA) block. Notably, ultrasound waveinitiated RAFTPISA can be used for expanding the scope of functional materials.
For another, Thang et al. adopted the ultrasound (990 kHz) to prepare polymeric NPs with various morphologies (Fig. 5A) [98]. The work first prepared vesicle morphology by Sono RAFTPISA (Fig. 5B). For comparison, thermalinitiated RAFT PISA was also investigated. Compared to thermal initiation, polymeric vesicles were formed at shorter PHPMA length by ultrasoundinitiated RAFTPISA. In addition, the size of obtained vesicles prepared by ultrasoundinitiated RAFTPISA was much smaller (126.2 nm versus 599.2 nm), and the PDI was also narrower (0.02 versus 0.23). However, worm structures were difficult to achieve due to high ultrasonic frequency. To overcome the influence of acoustic streaming effect, the core crosslinking (CCL) strategy was used as a solution to prepare wormlike nanoassemblies (Fig. 5C).
Ultrasoundinitiated RAFTPISA is emerging as a promising technique to fabricate NPs on a large scale. More importantly, the water in the system is used not only as a solvent but also as an initiation source. Therefore, no external initiators and additives are needed, which makes the ultrasoundinitiated RAFTPISA technique quite "green. " Compared with thermal initiation and photoinitiation, ultrasoundinitiated RAFTPISA does not suffer from uneven heating and insufficient depth of light penetration. However, the effect of high ultrasonic frequency on the morphol ogy of nanoassemblies during the PISA process still needs fur ther studies.

ATRP-PISA
ATRP is one of the most welldeveloped controlled polymeri zation techniques [99,100]. Compared with other polymeriza tion techniques, the unique characteristic of ATRP is the requirement of transition metal catalyst. In addition, the polymerization condition is mild without high temperature and pressure. ATRP can be conducted in various single or mixed solvents, such as MeOH, water/MeOH, and supercritical car bon dioxide (scCO 2 ) [101]. However, according to amounts of the published work, ATRPPISA does not seem to be a good PISA formulation, mainly because transition metal complexes can bring potential toxicity into polymeric NPs and unpleasant control. The first ATRPPISA work was reported by Pan and coworkers in 2007 [102]. What is more, after the reaction, the remaining metal catalysts in the nanostructures are harmful for use, especially in biomedical application [103]. How to improve and optimize the reaction condition of ATRP is the key issue in recent years.
Notably, initiators for continuous activator regeneration atom transfer radical polymerization (ICAR ATRP) is a powerful method to improve the reaction process by lowering the concen tration of metal catalyst. In 2016, ICAR ATRPPISA was first used to prepare poly(oligo(ethylene oxide) methyl ether meth acrylate)bpoly(benzyl methacrylate) (POEOMA-b-PBnMA) with the parts per million (ppm) concentration of copper catalyst ( Fig. 6A) [104]. Here, Matyjaszewski and coworkers used the CuBr 2 /tris(pyridine2ylmethyl)amine (TPMA) complex as cat alyst at RT or at 65 °C with various radical initiators. In detail, the research studied the effect of the catalyst concentration, solid content, and temperature on the selfassembly behavior. Notably, the glass transition temperature (T g ) of the coreforming block was 54 °C, which was between RT and 65 °C. Therefore, various assembly behaviors were observed. When at ambient temperature, large spheres were observed as the main morphol ogy. As the solid content and the DP of PBnMA increased, a fractaltype connectedbead structure was finally obtained (Fig.  6B). However, when the temperature was 65 °C, the morphol ogy transition from spheres to worms also appeared at low solid content (10%, w/w) (Fig. 6C). The reduced catalyst concentration in ATRPPISA can expand the scope of the relevant applica tions. More recently, Wang's group explored the morphological evolution of NPs using mixed POEOMA 24 and POEOMA 78 as macroinitiator [105]. Other polymers such as methoxypoly(eth ylene oxide)polyacrylonitrile (mPEO 113 PAN) was successfully synthesized via ICAR ATRP [106]. Spherical and wormlike NPs could be obtained through selfassembly. The reduced cat alyst concentration in ATRPPISA can expand the scope of the relevant applications.
In addition, activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) is another method to achieve ATRP process with lower concentration of catalyst (ppm) [107,108]. In such a process, highvalent transi tion metal complexes were used as catalysts and excess reducing agents were added intermittently to continuously reduce the highvalent transition metals to lowvalent oxides to effectively reduce the amount of catalyst. For instance, Matyjaszewski and coworkers reported the fabrication of crossedlinked NPs in ethanol by ARGET ATRPPISA [109]. Here, POEOMA with a terminal bromide was used as macroCTA to extend with gly cidyl methacrylate (GMA) or BnMA monomers. The different conversion rate between double bond and oxirane ring in GMA guaranteed the PISA process and the subsequent in situ cross link of NPs.
Organic-inorganic materials have been widely applied in catalyst, imaging, and nanomedical fields. To realize practical application of ATRPPISA, a novel NP consisting of POEOMAb-poly (2(perfluorohexyl)ethyl methacrylate (PHFEMA)-co-GMA) had been prepared by ATRPPISA [110]. Afterward, mercaptosuccinic acid was used to react with the epoxy group on the GMA, and the carboxyl groups were success fully introduced into the core of polymeric NPs. Subsequently, the nanoobjects can be used as template to prepare Fe 3 O 4 @ POEOMA organic-inorganic NPs. As characterized by TEM, dynamic light scattering (DLS), and thermogravimetric anal ysis (TGA), Fe 3 O 4 @POEOMA with an average diameter of about 150 nm was prepared. In addition, the diameter could be modulated by varying the packing parameter.
As known, the solvent, solid content, target DP, and temper ature are key factors on the morphology of the nanoassemblies prepared by PISA. Whether different PISA formulations can fabricate different morphologies with the same BCP composition is a question worth exploring. In 2017, Zhang and coworkers reported the fabrication of diblock copolymer poly(2hydroxy propyl methacrylate-b-benzyl methacrylate) (PHPMA-b-PBzMA) nanoassemblies to make a comparison between ATRP and RAFT [111]. The results indicated that both 2 robust polym erization techniques had good control on the MW and MW distribution. Notably, the ATRP dispersion polymerization was faster than the RAFT dispersion polymerization. In addition, nanoobjects prepared by ICAR ATRPPISA were larger than that prepared by RAFT polymerization.
More recently, the same group made another comparison between ATRPPISA and RAFTPISA [112]. The PEG-b-PS nanoassemblies were prepared by ATRPPISA and RAFTPISA. Although the targeted DP of PS was the same, the high copolymer dispersity existing in ATRPPISA led to various morphologies. In addition, the concentration of Cu salt on the effect of mor phology was explored. With the increase of Cu salt concen tration, the flowerlike structure was formed. As a robust PISA formulation, ATRPPISA can be used to prepare functional NPs on a large scale. Compared to RAFTPISA, ATRPPISA is usually performed at RT and does not involve the sulfur containing structure.

ROP-PISA
As mentioned above, nanoassemblies including spheres, worms, and vesicles can be prepared by controlled radical polymerization techniquemediated PISA. Nevertheless, the suitable monomers are mainly methacrylates and vinyls. However, the obtained NPs constructed by these PISA methods are usually not biodegradable. In this section, ROPPISA including ringopening metathesismediated PISA (ROMP PISA), ROP of Ncarboxyanhydrideinduced selfassembly (NCAPISA), and radical ROPinduced selfassembly (rROP PISA) will be elaborated carefully.

ROMP-PISA
ROMP is a nonradical method to achieve the polymerization process with Rubased catalysts under mild reaction conditions (Fig. 7). Cyclic monomers are used in the ROMPPISA process to fabricate polynorbornene (PNB)based NPs with various morphologies. In recent years, due to extraordinary functional group tolerance, ROMPPISA is emerging as a robust approach to prepare functional NPs under mild condition [113].
For example, Xie and coworkers first reported the facile preparation of functionalized polymeric nanoassemblies by  the ROMPPISA technique [114]. The homopolymer poly(2,3 dihydroxymethyl5norbornene) (PBNBE) was used to be chain extended with 7oxanorborn5eneexoexo2,3dicarboxylic acid dimethyl ester (ONBDM) monomers and selfassembled into NPs in toluene. Later, the same group reported the preparation of triblock copolymer PBNBE-b-poly(exoN(cinnamoyloxyethyl) 7oxanorborn5ene2,3dicarboximide) (PCONBI)-b-PONBDM with UVcrosslink core [115]. Through crosslinking strategy, fabricated NPs retained the original morphology in good sol vent as CHCl 3 , which indicated that the stability of obtained NPs had been improved greatly. Further, relevant works where higher order morphologies and functional NPs were generated were reported by other groups [116,117].
Further, more and more functional monomers have been used in ROMPPISA to achieve desired applications in recent years. Char and coworkers first reported the sulfurrich polymeric NPs constructed by ROMPPISA [118]. When increasing the ratio of the sulfurcontaining monomer during the PISA pro cess, the refractive index changed in a controlled manner, which could be used for monitoring the reaction process. In addition, enzymeresponsive polymeric NPs were prepared under opentoair condition [119]. First, l amino acidbased norbornene dicarboximide peptide substrate with sequence GPLGLAGGWGERDGS was chose to synthesize hydrophilic block, which was then chainextended with quaternary amine based phenyl norbornene dicarboximide to selfassemble into spherical micelles. The recognition sequence PLGLAG existing in the hydrophilic shell was the substrate of enzyme thermo lysin. Therefore, the obtained NPs would undergo phase tran sitions when exposed to the proteolytic thermolysin.
However, a major obstacle to conduct aqueous ROMPPISA is the poor solubility of Grubbs catalyst in water. One solution to make catalyst watersoluble is to modify it by introducing hydrophilic chain segment [120,121]. For this purpose, a single cationic quaternary amine Hoveyda-Grubbs secondgeneration initiator was used in the ROMPPISA to build welldefined NPs and detailed phase diagram was produced [120]. Recently, highly hydrophilic Grubbs catalyst was developed to obtain polymers with excellent control on MW and dispersity [122]. First, a short hydrophilic chain was polymerized in tetrahydro furan (THF) in the presence of G3. Afterward, the obtained macroinitiator could be chainextended with second hydro phobic monomers. During ROMPPISA, various morphologies were observed, such as spheres, worms, and vesicles.

ROP-PISA of NCA monomers
Polypeptidebased materials have been always attractive due to their distinct advantages including excellent biocompatibility and biodegradability [123,124]. Traditional selfassembly of polypeptides is generally conducted in a selective solvent, which is inefficient and timeconsuming. A facile onepot preparation strategy of polypeptide NPs was first reported by Du and coworkers in 2019 [125]. Here, PEG 45 NH 2 macroinitiator was used to polymerize with lphenylalanine NCAs (lPhe NCAs) in anhydrous THF (Fig. 8A). Compared with the above PISA formulations, this ROPPISA was conducted on the highly mild condition (10 °C) and the whole process did not need additional species. The hydrophilic/hydrophobic ratio and solid content could be finely regulated to achieve vesical morphology (Fig.  8B). Furthermore, the in vitro enzymatic degradation experi ments showed that most vesicles were degraded in 96 h when treated with trypsin (Fig. 8C). Reactive oxygen species (ROS) responsive methionine NCA monomer was also applied as hy dro phobic block to fabricate polymeric NPs under high solid content (up to 45%) [126]. The obtained polymeric NPs were incubated in different concentrations of H 2 O 2 (e.g., 0.1, 1, and 10 mM) to investigate their oxidative degradation behavior. Notably, these vesicles could be quickly degraded under various H 2 O 2 concen trations, which had great potential as intelligent nanocarriers. The novel NCAPISA brings great potential for biomedical appli cations including drug delivery and bioimaging.
More importantly, aqueous ROPPISA of NCAs, which was first reported by Bonduelle's group in 2019, is a big breakthrough because NCA monomers are believed to be moisturelabile [127]. To be specific, sodium bicarbonate aqueous solution (pH 8.5, 50 mM) was used to prevent the hydrolysis of NCAs and promoted the ROPPISA process. PEG5kNH 2 was used as the macroinitiator to be chainextended with γbenzylL glutamate Ncarboxyanhydrides (BLGNCAs) at 4 °C. The aqueous NCAPISA not only fabricated some conventional morphologies including spheres and worms but also obtained homogeneous needlelike structures. It was envisioned that this ROPPISA process was able to use different macroinitiators and NCA monomers. Notably, owing to the unique feature of polypeptides (e.g., secondary structure), NCAPISA is different from the above PISA formulations, which is also worthy of further exploration.
Very recently, the same group reported the effect of poly peptides' secondary structure on the morphologies of formed NPs in 2021 [128]. Two different monomers, i.e., BLGNCA and LeuNCA, that possessed αhelix and βsheet conforma tion, respectively, were used to investigate the influence of sec ondary structure on ROPPISA. The results demonstrated that the LeuNCA monomers enhanced the anisotropic character of the resulting selfassemblies. In fact, compared with αhelical polypeptides, βsheet polypeptides tended to form long rods with high aspect ratio. As a novel PISA technique, more and more research work is worth doing to explore the formation mechanism of NPs.
More recently, Du and coworkers carefully studied the effect of monomer chirality in chiralitycontrolled PISA by adjusting the chiral molar ratio of the Phe NCAs [129]. Various chiral com position also leads to different αhelix fraction and βsheet frac tion. When βsheet fraction increased to 50%, the uniform PISA behavior disappeared, which meant that the βsheet structure could weaken assembly behavior. Notably, in addition to mor phological control, by increasing of DPhe fraction, the biodeg radability of obtained NPs decreased. Adjustable biodegradation rate can be applied to control the drug release rate, thus having significant potential for biological applications.

rROP-PISA
Improving the biodegradability of polymers is of great sig nificance for bioapplications. Radical ROP is a method in which degradable groups (e.g., ester and thioester) can be introduced into the main chain of the polymer structure. Recently, Nicolas and coworkers first reported the rROPPISA based on cyclic ketene acetals (CKAs) in heptane, such as 2methylene4phenyl1,3dioxolane (MPDL) and 5,6benzo 2methylene1,3dioxepane (BMDO) [130]. As expected, the introduction of ester groups greatly improved the degrada tion property, which was verified by degradation test between the MPDLfree poly(lauryl methacrylate)-b-PBzMA (PLMAb-PBzMA) copolymer and PLMA-b-P(BzMA-co-MPDL) copolymers. Notably, by increasing the ratio of CKA, the better degradation could be achieved.
More recently, aqueous suspensions of degradable copolymer NPs were prepared by the 2step rROPPISA process [131]. First, poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) was chosen as solvophilic block to be chainextended with the 3 main CKA monomers [e.g., MPDL, BMDO, and 2methylene1,3dioxepane (MDO)] to in situ fabricate NPs in N,Ndimethylformamide (DMF). Then, the suspensions of NPs were dialyzed against water. To be specific, the POEGMA Biotoxicity of sulfur-containing species and high reaction temperature [130,131] chain preserved the stability of NPs when the formation was transferred from DMF to water. Notably, the longer POEGMA block provided better stability of NPs especially for high CKA contents. All these CKAcontaining NPs exhibited quick hydro lytic degradation under accelerated conditions (2.5 wt% KOH), proving that degradable vinyl polymeric NPs have great poten tial in bioapplications (Table 1).

Biomedical Applications
Nowadays, design and construction of biomedical nanoplatforms has gained great significance especially in the field of human healthcare. The stability of therapeutic agents can be greatly improved by encapsulation in NPs. Furthermore, by incorpo ration of targeting moieties, these NPs can efficiently target the disease sites. In recent years, PISA has been proved to be a robust technique to prepare functional NPs. Therefore, fabricating biomedical platform by PISA can not only achieve greater value of PISA but also improve the efficiency of the preparation [32,34,132]. So far, a range of polymeric NPs have been fabricated by PISA strategy for biomedical applications. In the following section, the efficient encapsulation and con trolled release of different cargoes including imaging agents, anticancer drugs, and enzymes will be demonstrated in detail (Table 2).

Bioimaging
The premise of precise treatment on diseases is able to pre cisely assess early diagnosis. Therefore, bioimaging has great significance on medical diagnosis and subsequent therapy. Current biological imaging techniques consisted of magnetic resonance imaging (MRI), computed tomography (CT), fluores cence imaging (FI), etc. [133]. Imaging agents are generally requested for precise detection of diseases. However, these small imaging agents usually have poor biodistribution and unsatisfied signaltonoise ratio. Notably, by encapsulation of these imaging agents in polymeric NPs, the above issues can be greatly alleviated.

CT imaging
As a common clinical imaging technique, CT has been widely used owing to its high efficiency [134,135]. However, the con ventional contrast agents (e.g., iohexol) are easy to be cleared quickly and distribute highly in normal tissues and organs, and iodinated NPs can overcome the above limitations. The most iodinated NPs prepared by traditional selfassembly method cannot meet the requirements of clinical imaging owing to the complicated and low loading efficiency of iodine. To overcome above limitations, iohexol NPs (INPs) have been successfully fabricated by PISA in high concentration (up to 120 mg ml 1 ) [136]. Here, the macroCTA PEG5kS1dodecylSʹ(α,α′ dimethylα′′acetic acid) trithiocarbonate (PEG5kTTC) was chainextended with hydrophobic monomer HPMA to fabri cate polymeric NPs (Fig. 9A). Then, iohexol acrylate was used as crosslinking agent to achieve efficient loading. Notably, the loading efficiency of iohexol in these NPs reached 36.5%, endowing these INPs great potential in CT as contrast agent. The in vivo pharmacokinetic studies showed that INPs had a longer halflife compared with free iohexol and a higherlevel CT signal. Notably, the INPs had low accumulation in liver and renal tissues, resulting in low biological toxicity and extended retention time. As expected, the novel INPs had a significant accumulation in tumor rather than normal tissues, which greatly improved the sensitivity of CT imaging in cancer diag nosis. In addition, according to the 3(4,5)dimethylthiahiazo (zy1)2,5diphenytetrazoliumromide (MTT) assay in vitro, the viabilities of cells incubated with INPs were all over 90%, which showed high biocompatibility of INPs. Finally, by per forming CT scans on the animals treated with INPs, main organs (e.g., liver and kidney) and representative blood vessels (e.g., renal vein, renal artery, aorta jugular vein, and the left ventricle) could be imaged clearly (Fig. 9B). These above results proved the INPs could effectively improve diagnostic accuracy and reduce biotoxicity.

MRI imaging
In addition to CT imaging, MRI as another conventional imaging technique also requires contrast agents to enhance the image quality, because it is easily affected by the high background signal from water. To improve the imaging quality, a strategy designed to minimize an overwhelming background signal and largely pre pare polymeric NPs had been reported [137]. Briefly, 19  Consistent with previous studies, the wormlike nanostructure with higher aspect ratio had the highest cell uptake efficiency. However, the vesicle NPs were difficult to be uptaken by cells. The cell uptake result was also confirmed by confocal laser scanning microscopy (CLSM). All these results showed the fluorinated NPs with wormlike morphology had the potential for in vivo cell tracking and cell imaging. Furthermore, more fluorinated NPs can be constructed by PISA to perform bio imaging and disease diagnosis. More recently, 19 F MRI nanotracers were first fabricated by aqueous RAFTPISA, using N(2,2,2trifluoroethyl)acrylamide (TFEAM) as coreforming monomers (Fig. 10A) [138]. However, the PEGbPTFEAM NPs did not have strong 19 F NMR signal, which was attributed to the limited mobility of the coreforming blocks, further leading to the poor magnetic relaxation properties. Therefore, hydrophilic Nhydroxyethyl acrylamide (HEAM) monomer was used to improve chain hydration and mobility. With the increasing content of HEAM (up to 30%), the signal tonoise ratios (SNRs) of the NP dispersions gradually increased (Fig. 10B). In addition, for further biomedical application, ensuring the stability of the micelles, PEG 91 b(PTFEAM 320 statPHEAM 80 ) (F5H2) was used as nanotracer. In in vivo imag ing experiment, after the injection of F5H2 nanotracers into the right hind leg of mouse, obvious 19 F MRI signals with excel lent SNRs could be monitored, indicating that the novel fluori nated NPs constructed by PISA had great potential in biomedical imaging (Fig. 10C).

Fluorescence imaging
In addition, fluorescent materials have been developed and grown vigorously in recent years owing to the great potential in biomedical fields [139][140][141]. Some organic fluorescent molecules are limited in the bioapplication, ascribed to poor solubility in water. RAFTPISA is a robust technique to pre pare fluorescent NPs for bioimaging.
For instance, chalconecontaining NPs with uniform spherical morphology had been successfully prepared by using chalcone derivative terminated with ethylene group [142]. Interestingly, when the NPs were transferred from dioxane to water, the size shrank distinctively from ~200 nm to ~100 nm. Furthermore, the emission intensity significantly improved, which was con tributed to the aggregationinduced emission feature of chalcone. The biocompatibility of the fluorescent NPs was characterized by CCK8 kit assay. The cell still maintained high cellular activity (above 90%), although the concentration of NPs reached 40 μg/ml. Further, HeLa cells were cultivated with fluorescent NPs (10 μg/ml) for cell imaging. The obtained fluorescent images with high contrast confirmed the great potential of fluorescent NPs in cell imaging.
More recently, poly ((oligo(ethylene glycol) acrylate)-bpoly(benzyl acrylate) (POEGA-b-PBzA) NPs had been fabri cated by photomediated RAFTPISA (Fig. 11A) [143]. Notably, sub50nm spherical NPs were successfully obtained by the adjustment of chain length (Fig. 11B). Fluorescent molecules were loaded into the NPs through facile swelling procedure. Attractively, the fluorescent sub50nm NPs had quantum yield higher than 50% in various solvents. Besides, characterized by the cytotoxicity investigations and the preliminary cell uptake assay, these NPs possessed high biocompatibility and great potential for bioimaging. Confocal fluorescence microscopy images showed that various cells had fluorescent signals after being incubated with the NPs, and these imaging results by NPs and cell tracker were consistent with each other, which proved the feasibility of the fluorescent NPs in biological imaging (Fig. 11C). Notably, although the concentration of dye was 15 nM in water, the higher intracellular fluorescence could be detected. Moreover, the process of polymeric NPs penetrating across cell membrane was observed, and the fluorescence inten sity could be monitored within 22 s after injection. In addition, cytoplasmic vesicles (e.g., endosomes and lysosomes) had a higher fluorescence intensity compared with cytosol and nucleus.
What is more, azoreductases are commonly found in the human colon, which drives azobenzenebased polymeric NPs to apply in colon disease treatment. A series of tetraphenyleth ylene (TPE)azobenzene fluorescent probeincorporated NPs had been fabricated by RAFTPISA, while doxorubicin (DOX) was encapsulated in the selfassembly process. Then, the obtained NPs were used for bioimaging and treatment (Fig. 12A) [144]. When these NPs were exposed to azo reductase, the azo double bond would be destructed, leading to the disassembly of NPs and further release of DOX. Notably, the cumulative release of DOX reached 58.5% at 24 h. Instead, only a very small per centage (<10%) of DOX was released without the presence of azoreductase. In addition, the cleavage of azo bond eliminated the fluorescence resonance energy transfer (FRET) process. Therefore, a gradual increase in luminescence intensity was observed within 24 h. The DOXloaded micelles were first incu bated in the presence of enzyme azoreductase for 24 h and then used in cell uptake experiment. After 4 h of incubation, the DOX fluorescent signal was gradually enhanced, indicating that DOX successfully entered cell nuclei and achieved accumulation (Fig.  12B). The obvious fluorescent signal from TPE fragment was also observed after 4 h of incubation. Notably, the obtained NPs with integrated diagnostic and therapeutic functions showed great potential in biomedical applications. Similarly, a novel near infrared (NIR) fluorescent nanoprobe was designed to realize synchronous drug release and cell imaging [145]. The NIR fluorophore AzaBODIPY moiety was introduced into the hydrophilic macro CTA and chainextended with the hydro phobic monomer BzMA to fabricate spherical and vesicular NPs. In a simulated colon environment, reductiontriggered drug release and realtime fluorescent behavior of these obtained NPs were investigated. With the extension of incubation time up to 24 h, the gradual release behavior of DOX (~60%) was observed. In the process of incubation, the NPs gradually dis sociated; thus, the aggregationcaused quench (ACQ) effect disappeared and increased fluorescence intensity was observed. Then, the fluorescent NPs were incubated with mouse colon cancer cells (CT26 cells) under normoxia (16% O 2 ) and hypoxia (1% O 2 ) to test cell imaging. Compared with the normoxic envi ronment, higher fluorescence intensity was observed in the hypoxic environment, which could explain the overexpression of azoreductases in hypoxic cancer cells. In general, the NIR fluorescent nanoprobes can be used to build an integrated plat form for diagnosis and treatment.
In conclusion, polymeric NPs constructed by the PISA method have been used in bioimaging. However, owing to the strict requirements for solvents and monomers, current NPs are far from satisfaction. Furthermore, more suitable PISA formulations to improve the biocompatibility of NPs are also urgent. In fact, the significant values of encapsulation of imaging agents for bioimaging are worthy of more research work.

Disease treatment
As commonly used cancer treatment, chemotherapy, where small molecule drugs kill cancer cells, usually causes serious side effects to patients. Meanwhile, the nonspecific distribution of therapeutic drugs is another obstacle. Nowadays, nanocarriers have been widely used to improve the solubility and pharma cokinetics of therapeutic drugs. Generally, extended blood cir culation time and targeted accumulation of these drugs can be achieved by encapsulation in polymeric NPs. As mentioned, PISA is emerging as a robust technique to fabricate suitable nanocarriers for encapsulation of therapeutic drugs. Plenty of NPs have been exploited for intracellular delivery [146][147][148][149], proving that PISA has great potential in constructing drug deliv ery systems (Fig. 13).

Small drug delivery system
For example, poly[oligo(ethyleneglycol) methacrylate]b[poly(sty rene)-co-poly(vinyl benzaldehyde)] [POEGMA-b-P(ST-co-VBA)] NPs with various morphologies were prepared by RAFTPISA [150]. DOX was successfully encapsulated in the core of NPs through conjugation with aldehyde groups via pHbreakable bonds, and the drug loading efficiency reached 67%. Most drugs (~80%) could be released from nanocarriers at pH 5.0 in a con trolled manner, which was much higher than that at pH 7.4. Subsequently, the cellular uptake behaviors of these DOXloaded NPs were investigated by incubating with breast cancer cells (MCF7) for 24 h. Notably, during the incubation process, the high est cell uptake efficiency was observed for worm and rodlike structures by using flow cytometry. The final result could explain that the higher aspect ratio of worm and rodlike structures caused a greater adhesion of polymeric NPs to the cell membrane com pared with vesicle and spherical micelles. In addition, the half max imal inhibitory concentration (IC 50 ) of the worm and rodlike structures was 7 times lower than spherical micelles. The above result was consistent with the higher cell uptake of the worm and rodlike NPs. Similarly, Pan and coworkers also explored the influ ence of morphology on drug delivery processes [151]. Briefly, poly((N,Ndimethylamino)ethyl methacrylate) (PDMAEMA) was used as macroCTA to polymerize with p(methacryloxyethoxy) benzaldehyde (MAEBA) units for in situ fabrication of polymeric NPs. The aldehyde groups of PMAEBA were used to conjugate with DOX. Afterward, HeLa cells were incubated with these NPs with various morphologies (e.g., spheres, nanorods, and vesicles) for 24 h. The results by testing the fluorescent intensity of DOX indicated that the cellular uptake rate had the following order: vesicle > nano rod > sphere. However, the in vitro cytotoxicity of DOXloaded nanorod was more cytotoxic than the DOXloaded vesicle, which demonstrated that more DOX molecules were released from the DOXloaded nanorods. Relatively more nanorods than vesicles were localized in acidic organelles, and the drugs were more likely to be released in acidic conditions. These above results may guide the future design and fabrication of drug delivery nanosystem and improve the effectiveness of the disease treatment.
As known, drug loading efficiency is a key parameter for the encapsulation of drugs. Recently, the introduction of coordi nation interactions between polymers and chemo drugs is an efficient strategy to achieve high drug loading efficiency [152]. In addition, design and fabrication of prodrug monomers is also an ideal strategy to improve drug loading efficiency [153][154][155]. During PISA, therapeutic prodrugs can be directly conjugated to polymeric backbones. For example, the camptothecin (CPT)containing copolymer PEG-b-P(2(2methoxyethoxy)  ethyl methacrylate PEGbP(MEO 2 MA)-co-CPTM) was used as macro RAFT agent to polymerize with BzMA monomers to achieve prodrug NPs [154]. It was worth mentioning that this method realized polymerization, selfassembly, and drug load ing simultaneously. Therefore, the obtained prodrug NPs did not need complex purification, saving a lot of time and labor. Notably, the drug content could be facilely tuned by adjusting the molar ratio of CPTM/MEO 2 MA. Notably, encapsulated CPT could be released under reductive environment. The obtained prodrug NPs were conducted with in vitro drug release at different concentrations of glutathione (GSH) (0.01, 5, and 10 mM). The higher cumulative CPT release was achieved with the higher GSH concentration. The cumulative CPT release reached 42% after incubation in 10 mM GSH for 48 h. Notably, the obtained polymeric NPs without CPT conjugation were noncytotoxic even when the concentration was up to 1,000 μg/ml. To further study the intracellular drug release of the prodrug micelles, CLSM results demonstrated that the CPT drug was gradually released and accumulated in cell nucleus. In general, PISA provides a robust method to con struct CPT prodrug NPs, facilitating the development of the field of nanodrug delivery.
Recently, to overcome the disadvantages of small conven tional platinum analogs and the limitations of traditional assembly methods, one approach was used to use ROMPPISA in water to incorporate platinumbased drug into polymeric NPs [156]. In brief, the poly(oligo ethylene glycol) (POEG) stabilizer was directly chainextended with the cisplatin analog norbornene dicarboximide (CAND) for the fabrication of drug loaded NPs. Meanwhile, pHresponsive 2(diisopropylamino) ethyl norbornene dicarboximide (DEND) monomers were used to achieve controllable disassembly upon acid tumor envi ronment. The effect of the size and zeta potential on cell uptake procedure was systematically studied. Among these platinum loaded NPs, smaller NPs had higher cytotoxicity, while posi tively charged NPs had higher cell uptake efficiency than the negatively charged ones, facilitating the design of NPs with better performance. In addition, owing to the ability of preparing platinumloaded NPs in high concentration, ROMPPISA promises to formulate pharmaceuticals for low volume injec tions at high dose for human disease.

Macromolecular drug delivery system
Besides small therapeutic drugs, proteins are specific and highly active substances, which play an important role in regulation of vital activities. In recent years, much effort has been devoted to deliver therapeutic proteins for precise nanomedicine. However, owing to its poor stability and short circulation time in vivo, the application prospects are greatly limited.
The use of therapeutic proteins in the clinic has been increas ing quickly in the past few years because of high efficiency [157,158]. Unfortunately, the stable delivery of proteins is quite difficult because proteins are susceptible to immune responses and are easily inactivated upon internal or external conditions. Normally, modification of proteins by using hydrophilic chain segments can extend their in vivo blood circulation time. For example, the PEGylated interferonα (PEGASYS) has 8.6fold halflife time than native therapeutic protein interferonα (IFN). What is more, Gao and coworkers improved the in vivo halflife time of IFN as long as 83.8 h by sitespecific in situ PISA (SIPISA) strategy (Fig. 14A) [159]. However, PEGylation products usually have lower bioactivities owing to the presence of positional isomers. Here, to avoid creating positional iso mers, POEGMA was used as an alternative to PEG. Compared with IFN, the plasma IFN levels of IFNmicelle consisted of IFNPOEGMAPHPMA decreased slowly after intravenous injection. Then, the biodistribution of IFNmicelle was studied in the ovarian tumorbearing mouse model. The IFN levels of IFNmicelle had significantly increased accumulation in major organs compared with PEGylation and POEGMA conjugation of IFN. Furthermore, the IFNmicelle was more effective than PEGASYS in terms of inhibiting tumor growth (Fig. 14B). More significantly, after treatment with IFNmicelle, the survival rate of mice remained 100% as long as 120 days (Fig. 14C). The above results indicated that the SIPISA has great potential to improve the pharmacology of therapeutic proteins.
However, the chemical modifications of proteins may cause some changes to their inherent characteristics. Therefore, physical encapsulation of the protein is also a good choice. Traditional method of encapsulating therapeutic protein usually requires multiple steps, and the polymeric vesicles need to be permeable. lAsparaginase (ASNS) is a therapeutic enzyme used for leu kemia treatment, which has been encapsulated in vesical cavity by the onepot method [160]. The successful preparation of ASNSloaded vesicles by aqueous photoPISA has been charac terized by TEM, atomic force microscope (AFM), and DLS meth ods. The mild reaction condition of photoPISA did not disrupt the structure of proteins. A greater stability in vitro and in vivo had been achieved compared to native protein or PEGylated con jugate. Besides, the antibodies would be blocked from entering the vesicle. Thus, the immunogenicity of the enzyme was reduced under the protection of vesicles, lowering the risk of an immune response. Notably, the in vitro experiments showed that cell pro liferation suffered from enhanced inhibition when treated with ASNSloaded vesicles (58%) compared to PEGASNS (61%) and free ASNS (78%). Finally, ASNS encapsulated in the polymeric vesicles has lower accumulation in the liver and the kidneys com pared to free ASNS. Therefore, the sizeselectively permeable vesicle prepared by PISA is a good choice to encapsulate protein without chemically altering the protein, thus providing better therapeutic effect.
More recently, an innovated dengue virusmimicking tri block vesicle was designed to treat triplenegative (TN) breast cancer [161]. Here, poly(glycerol monomethacrylate) (PGMA) as macroCTA was successively chain extended with HPMA and 2(diisopropylamino)ethyl methacrylate (DPA) mono mers. The protonation behavior of PDPA block under acid environment endowed the vesicles with ultra pHresponsive behavior. To achieve active targeting property, a small fraction (3 mol%) of the PGMA was replaced by poly(2(methacry loyloxy)ethyl phosphorylcholine) (PMPC), which could actively target SRB1 scavenger receptoroverexpressed TN breast can cer cells. In cell uptake experiments, when treated with vesicles without PMPC block, all cells showed low uptake. However, triblock copolymer vesicles containing PMPC block could actively target TN breast cancer cells. The obtained vesicles were used to load plasmid DNA (pEGFP) by electroporation and then used in the cell uptake experiments to prove the ability to target the nuclear region. By characterizing the expression of fluorescent EGFP within the cells, the vesicle could deliver func tional biomolecules (e.g., plasmid DNA) to the nuclear region. Finally, the bionic vesicles constructed by PISA can provide better therapeutics for TN breast cancer.
What is more, heparin is a sulfated polysaccharide, having great potential in wound healing and tissue regeneration by stabling the fibroblast growth factors (FGFs). Notably, PISA can also be used to fabricate heparinmimicking sulfonated NPs.
For this case, poly(2acrylamido2methyl propane sulfonic acid) [P(AMPS)] was used as macroCTA to chainextend with St units [162]. The NPs showed low biotoxicity to murine embryonic fibroblast cells even if the concentration reached 1 mg ml −1 . Also, the heparinmimicking NPs displayed greater cellular proliferation than heparin (400% versus 150%). The difference of cellular proliferation may be mainly attributed to the existence of numerous surfaceactive P(AMPS) chains at the surface of NPs, thus promoting the stabilization of FGF. In general, PISA provides a facile route to fabricate sulfonated NPs at high solid content, thus facilitating the study of heparin mimicry.

Biocatalysis
Enzymes have received significant attention for the high effi ciency in biocatalysis [163]. However, enzymes are prone to be inactivated as a result of being stimulated by external environ ment. The nanoreactor can isolate the protein from the external environment and enable the protein to interact with small substrates simultaneously. In 2017, O'Reilly and coworkers reported the polymeric vesicles consisting of PEG-b-PHPMA prepared by aqueous RAFT photoPISA (Fig. 15A) [164]. In the paper, the permeable nature of the PHPMA membrane was discussed for the first time. The PHPMA membranes were highly hydrated; thus, small molecules could get in and out of vesicles, while the enzymes were confined in the cavities. A range of functional enzymes, such as HRP and GOx, had been successfully encapsulated into polymeric vesicles, and the enzymes also remained active owing to the permeability of the membrane. GOx consumed dglucose, simultaneously produc ing δglucono1,5lactone and H 2 O 2 as accessory substance. Then, the produced H 2 O 2 was used to catalyze the oxidation of 3,3′dimethoxybenzidine (DMB) to a colored dimer product by HRP. Only when both enzymes and reagents existed, cascade reaction can be observed (Fig. 15B). Furthermore, cascade reaction between HRP and GOx proved the permeability of the PHPMA membrane.
Later, the same group adopted the crosslinked strategy to adjust the membrane permeability of nanoreactors. First, welldefined enzymeloaded vesicle that selfassembled by PEG-b-P(HPMA-co-GlyMA) BCPs was prepared at ambient reaction condition by photoPISA [165]. The pendant epoxide groups of PGlyMA units in the hydrophobic layer provided the opportunity to react with amino groups to achieve crosslinking effect. Next, various kinds of primary amines, such as PEG diamines and linear aliphatic diamine, were used to cross the membranes of these vesicles. However, the obtained enzyme loaded vesicles all showed decreased enzyme activity, which was ascribed to decreased membrane permeability. Among them, the vesicle crosslinked by hydrophobic amines could reduce permeability up to 80%. In general, the increased thickness and reduced permeability as a result of membrane crosslinking adjusted the enzyme activity by controlling the diffusion process of substrates. This study provided a robust strategy to tune the membrane permeability, which may have irreplaceable guiding significance on the future design of nanoreactors.
Although changing the DP of hydrophobic block or copo lymerizing with other monomers can adjust the membrane permeability, the vesicle morphology may also change. Recently, welldefined copolymer vesicles with tunable membrane thick nesses and compositions prepared by aqueous seeded photoPISA were used as enzymatic nanoreactors [166]. By copolymerizing with HPMA or other functional monomers, vesicles with var ious compositions and membrane thicknesses could be suc cessfully fabricated, without changing the vesicle morphology. Notably, given the sizeselective permeability of the PHPMA membrane, the HRPloaded vesicles could oxidize 2,2′azinobis (3ethylbenzothiazoline6sulfonic acid) (ABTS) into ABTS + in the presence of H 2 O 2 . By tuning membrane thickness or hydrophobicity, the enzymatic reaction rate could be finely adjusted.

Antimicrobial
In recent years, the number of novel antibiotics entering the clinic is gradually decreasing, and the threat of antibiotic resist ance is gradually increasing. Based on the forecast, the bacterial infections will become the leading cause of death in the near future [167,168]. Therefore, novel and effective antimicrobial agents have been explored to fight against bacterial infections. Therein, combining antimicrobial enzymes and polymeric nan oreactors is a potential method to overcome microbial infec tions. In the past, honey was used as natural antimicrobial medicine, which is contributed to the H 2 O 2 generated by GOx in honey. Encapsulation of GOx into polymeric nanoreactors can maintain the activity of enzyme, thus providing antibacte rial properties. The PEG 113 bPHPMA 400 vesicles were prepared by thermalinitiated aqueous RAFTPISA at 37 °C, and GOx was encapsulated during the assembly process with an encap sulation efficiency of about 24% (Fig. 16A) [169]. The catalytic activity of nanoreactors was characterized under various glu cose concentrations, in which higher glucose concentration produced more H 2 O 2 . Staphylococcus aureus and Staphylococcus epidermidis were chosen to investigate the antimicrobial prop erties of GOxloaded nanoreactors. When incubated with the GOxloaded nanoreactors for 24 h, the growth of 2 Grampositive staphylococcal strains was inhibited (Fig. 16B). According to colonycounting assay, the GOxloaded nanoreactors reduced bacterial growth by 5 logs for S. aureus and 6 logs for S. epidermidis in the presence of 170 mg l −1 glucose concentration. Furthermore, the antibacterial universality of GOxloaded nanoreactors was also investigated by conducting antibacterial tests with Escherichia coli and Klebsiella pneumoniae. Notably, the growth of K. pneumoniae could be reduced at 800 mg l −1 glucose con centration. However, the reduced growth of E. coli was achieved at 5,170 mg l −1 glucose concentration, which was far higher than the normal glucose concentration in human body. In gen eral, the antimicrobial property of GOxloaded nanoreactors was highly effective for most strains of bacteria. In addition, in view of the sensitivity of fibroblasts to H 2 O 2 , the concentration of GOxloaded nanoreactors could be decreased to 0.69 mg ml −1 (Fig. 16C).
As known, short cationic peptides can combine with phos pholipid headgroups on the bacterial membranes and further promote cell death. Therefore, most antimicrobial peptides (AMPs) are cationic peptides. Similar to small molecule drugs, AMPs also suffer from poor stability, salt sensitivity, and toxicity. Therefore, AMPcontaining NPs can be prepared by PISA to overcome the above limitations. The polylysine macroCTA was chainextended with HPMA monomers under aqueous condition to prepare NPs with various morphologies [170]. Furthermore, the various NPs were used to investigate the anti microbial property of Grampositive bacterium. The experi mental results showed that the spherical NPs consisting of polylysinebPHPMA 12 had a 99.96% bacteria removal, which was significantly high compared with worms and vesicles. The higher AMP content and the larger specific surface area of spherical NPs were the main reasons for the result. Notably, the polylysinebPHPMA 12 NPs could be used to prepare film membranes, and then the membranes were applied to filter the water containing S. epidermidis.

Conclusion and Future Perspective
Over the past decade, with the development of polymerization technology, a range of monomers can be applied to PISA for the preparation of NPs. Notably, PISA has shown many advan tages in fabrication of various nanoplatforms especially in terms of high efficiency and laborsaving. Among the various PISA formulations, RAFTPISA is mostly investigated and used. The related reaction mechanism and morphology control have been thoroughly studied. However, nondegradable carbon-carbon skeletons and the biotoxicity of thioester func tional groups are not good for biomedical application. Other PISA formulations (e.g., NMPPISA and ATRPPISA) can pro vide complement to RAFTPISA. Especially, NCAPISA is an emerging PISA approach. Considering facile synthesis and excellent biocompatibility of polypeptides, some pioneering work has already been reported. The biomedical nanoplatforms fabricated by PISA have been used in the field of bioimaging, disease treatment, biocatalysis, and antimicrobial. As a robust technique to scale up preparation of NPs, PISA is able to encap sulate functional molecules for various applications. However, the development of functional nanoplatforms by PISA is still in its infancy, and many issues should be solved in the future. 1. The types of monomers suitable for PISA are worthy of more exploration. In recent years, with the development of controllable polymerization technology, more monomers can be used in PISA formulations. However, compared with tradi tional selfassembly method, PISA formulations should con sider the solubility of the monomer and the choice of solvent, which resulted in a relatively small number of suitable mono mers. More specifically, a range of functional BCPs have been designed to construct nanoplatforms by traditional selfassembly. In comparison, the nanoplatform constructed by PISA still needs further improvements. Therefore, it is necessary to expand the scope of monomers. In consideration of the high efficiency of PISA method, more efforts should be used.
2. Current biomedical nanoplatforms constructed by PISA are relatively simple and very limited. More biologyrelated experiments should be conducted to prove the great potential of PISA in fabricating biomedical platforms. In additional, most of the studies are based on RAFTPISA, which may bring bio toxicity into polymeric nanoplatforms. There are convenient ways to eliminate the end groups of CTAs, which undoubtedly introduce complexity into the PISA process. Notably, emerging NCAPISA that has both high biocompatibility and mild prepa ration is extraordinary. Therefore, exploring more NCA monomers that have stimuliresponsive characteristics can be taken into account and may boost future development of PISA.
3. The encapsulation efficiency of functional molecules by PISA is still challenging. For biomedical applications, the current approach is to physically encapsulate these molecules in the cav ities of fabricated vesicles. However, the encapsulation efficiency is unsatisfactory and the purification is complex. In addition, the release of encapsulated molecules is also not controllable in most PISA instances. In fact, pH response, enzyme response, light response, magnetic response, and ultrasonic response can be introduced into the PISA system to control local release. Therefore, designing prodrug monomers or conjugating func tional molecules to monomers via stimuliresponsive linkages are worth considering. This strategy not only improves the encapsulation efficiency but also allows for controlled release.
4. The broad application of PISA needs to be further explored. In addition to its biomedical application, PISA can also be applied in the field of nanocomposites, functional coatings, hydrogel, and so on. PISA technology has a signif icant potential in a variety of application areas. Although the PISA technology has been developed for many years, its application is still in the exploration and preliminary stage. Therefore, more effort should be put to enrich the applica tion of PISA.