Nanogel engineering for new nanobiomaterials: from chaperoning engineering to biomedical applications

The Chemical Record, Vol. 10, 366–376 (2010) © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.366 www.tcr.wiley-vch.de

Nanogel Engineering for New Nanobiomaterials: From Chaperoning Engineering to Biomedical Applications

YOSHIHIRO SASAKI AND KAZUNARI AKIYOSHI*Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,2-3-10, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan

Received 24 March 2010

ABSTRACT: Nanosize hydrogels (nanogels) are polymer nanoparticles with three-dimensional networks, formed by chemical and/or physical cross-linking of polymer chains. Recently, various nanogels have been designed, with a particular focus on biomedical applications. In this review, we describe recent progress in the synthesis of nanogels and nanogel-integrated hydrogels (nanogel cross-linked gels) for drug-delivery systems (DDS), regenerative medicine, and bioimaging. We also discuss chaperone-like functions of physical cross-linking nanogel (chaperoning engineering) and organic-inorganic hybrid nanogels. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 10: 366–376; 2010: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.201000008

Key words: nanogel engineering, drug-delivery system, chaperoning engineering, physically cross-linked nanogels

T H E C H E M I C A L

R E C O R D

1. Introduction

Nanogels are nanometer sized hydrogel nanoparticles (<100 nm) with three-dimensional networks of cross-linked polymer chains. They have attracted growing interest over the last several years owing to their potential for applications in biomedical fi elds, such as drug delivery systems (DDS) and bioimaging. Usual polymer nanoparticles, such as nanospheres, have a densely packed polymer inside the core structure. In contrast, nanogels are able to stably trap bioactive compounds such as drugs, proteins, and DNA/RNA inside their nanospace with polymer networks. Moreover, nanogels show a rapid response to microenvironmental factors such as temperature and pH because of their nano-scaled dimension. The proper-ties are useful for the controlled release of bioactive com-pounds. Nanogels have been prepared using various methods, which can be classifi ed into two categories according to their cross-linking structure: chemically (covalent) cross-linked nano-

gels which form cross-linking points by covalent bonds, and physically cross-linked nanogels with non-covalent bonds, such as hydrogen bonds, electrostatic and hydrophobic interac-tions. Kabanov and colleagues reported the fi rst chemically cross-linked nanogels made from poly(ethylene glycol) (PEG) and polyethylenimine (PEI) for the delivery of antisense oligonucleotides.1,2 Our laboratory reported the fi rst physically cross-linked nanogels using self-assembly of cholesterol-bearing polysaccharides in water through the study of self-organization of amphiphilic polymers.3 We also applied the physically cross-linked nanogels as nanocarriers in develop-ment of DDS.4 Recently, several review articles regarding

� Corresponding author: Tel: +81-3-5280-8020, FAX: +81-3-5280-8027,E-mail: [email protected]

N a n o g e l E n g i n e e r i n g f o r N e w N a n o b i o m a t e r i a l s

www.tcr.wiley-vch.de 367The Chemical Record, Vol. 10, 366–376 (2010) © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

� Kazunari Akiyoshi has been a Professor at the Department of Polymer Chemistry, Graduate School of Engineering at Kyoto University (Japan) since September 2010. He was born in Tokyo, Japan, in 1957. He received his PhD degree at Kyushu University (Japan) in 1985. After post-doctoral research for 2 years at Purdue University (USA) with Professor Ei-ichi Negishi, he joined Faculty of Engineering in Nagasaki University (Japan) as a lecturer. In 1989, he moved to Kyoto University as an Assistant Professor and was promoted to Associate Professor in 1993. In 2002, he moved to the Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental Uni-versity as a Professor. He was a visiting Associate Professor of Luis Pasteur University (France) (1997) and Precision and Intelligence Laboratory, Tokyo Institute of Technology (Japan) (2005–2007). He received the Award of the Society of Polymer Science, Japan (1997) and 2001 Barre Lecturer Awards, the University of Montreal, Canada (2001). His scientifi c interests are focused on developments of bio-inspired nano-organized systems for new biomaterials (polysac-charide-, nanogel-, and liposome-engineering) and utilization in biotechnology and medicine, such as drug delivery system or tissue engineering. �

� Yoshihiro Sasaki was born in Nagoya, Japan, in 1972. He graduated from the Faculty of Engineering, Kyoto University in 1995, and obtained his PhD in 1999 from the same university, for his work on polymer chemistry under the guidance of Prof. J. Sunamoto. After his PhD, he worked as an Assistant Professor at Nara Institute of Science and Technology, working on artifi cial cell membranes for application to nano-bioscience. In 2003, he performed a short stay as a visiting scholar at the University of Notre Dame, U.S.A. and joined Prof Akiyoshi’s group at the Tokyo Medical and Dental University in 2008. His research covers a broad range of topics in bioinspired chemistry and nanobioscience centered around supramolecular chemistry, focusing on artifi cial cells (liposome), organic-inorganic nanohybrids, self-assembled nanogels, synthetic receptors, and molecular devices as well as the study of their biomedical applications including gene delivery and cancer chemotherapy. �

biomedical applications of nanogels have been published, with a particular focus on DDS.5–7 In this article, we fi rst overview the recent progress of nanogel synthesis, then focus on physi-cally cross-linked nanogels and nanogel-based biomaterials. We also discuss the design of an artifi cial chaperone (chaperon-ing engineering) and organic-inorganic hybrid nanogels.

2. Chemically Cross-Linked Nanogels

2.1 Nano- or Micro-Emulsion Polymerization

Typically, chemically cross-linked nanogels are synthesized under diluted conditions using a cross-linking reaction of water-soluble polymers such as polysaccharides, which are modifi ed with reactive groups such as vinyl groups and thiol groups. To obtain nanogels with a well-controlled size, nano- or microemulsion polymerization methods have been widely

used.5,6 For example, nanogels were obtained by using polym-erization within the core of oil-in-water (O/W) nano- or microemulsions in the presence of surfactants (Figure 1a).6 By using an amphiphilic polyethylene glycol (PEG) macromer instead of the low molecular weight surfactant, a variety of functional nanogels enveloped in PEG chains have been syn-thesized.8 PEGylated nanogels with pH-responsive swelling and shrinking phase transitions have been applied to nanocar-riers and bioimaging.9,10

Inverse microemulsion polymerization using water-in-oil (W/O) emulsions has recently received research attention (Fig. 1b). In particular, monodispersed nanogels of various sizes have been synthesized using atom transfer radical poly-merization (ATRP) in W/O nanoemulsion.5 Water-soluble drugs including anticancer drugs (e.g., doxorubicin) and bio-active macromolecules (e.g., DNA and proteins) were easily incorporated into nanogels using this method.

The Chemical Record, Vol. 10, 366–376 (2010) © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

T H E C H E M I C A L R E C O R D

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2.2 Cross-Linking of Block Copolymer Micelle

Amphiphilic block copolymers are known to self-assemble monodispersive polymer micelles in water. Stable nanogels with a narrow size distribution were obtained by covalent chemical cross-linking of the hydrophilic or hydrophobic polymer chains in the polymer micelle (Figure 2). A variety of chemical cross-linking methods have now been developed, including carbodiimide-mediated amide bond cross-link-ing,11–13 quaternization of amino groups,14 “click” chemistry,15 and photo-cross-linking.16,17 In addition, redox-responsive nanogels can also be obtained using disulfi de bonds as cross-linking points.18 Nanocarriers with disulfi de bonds have been found useful in effi cient gene delivery.19 More recently, a novel method for the preparation of star-like nanogels has been reported, in which the core was cross-linked by bond exchange reactions of dynamic covalent bonds between two types of block copolymers with differing chain-lengths.20 Biomedical applications of pH-responsive or thermoresponsive nanogels have substantially progressed in recent years.

2.3 Nano Template Method

A new method has been developed to precisely control the size of nanogels using liposomes as a template (Figure 3a).21 Liposomes are spherical and closed structures formed by the self-assembly of amphiphilic lipid molecules in water.22 Nanogels can be obtained using the aqueous cores of the nano-sized liposomes as reaction vessels. Upon removal of the lipid molecules covering the hydrogel, nanogels that closely match

the size of liposomal template can then be obtained. Lipid-coating nanogels are also of interest as potential nanocarriers.

Hollow nanogels can be prepared using nanoparticles as a template. For example, gold nanoparticles were used as a template to grow a nanoscale polymer shell formed with hydrogel, which can be followed by removing the template to yield stable hollow nanogels (Fig. 3b).23 In another example of the nano template method, monodispersive silica nanoparticles were used to give hollow nanogels formed with poly(N-isopropylacrylamide).24 In this procedure, the size of the inner cavity is approximately the diameter of the solid nanopar-ticles used as the template. These nanogels are of particular interest because they also possess the ability to function as nanocapsules.

2.4 Top-Down Methods using Lithography

Defi ned molecular architecture on the nanoscale is important especially for the development of nanocarriers in DDS. PEG cross-linked nanogels were synthesized on silicon wafers using a top-down particle lithographic fabrication method called PRINT (Particle Replication In Non-wetting Templates).25 Monodispersed nanogels with various shapes have been obtained using the PRINT method.

The recent improvement to the PRINT method, the Step and Flash Imprint Lithography (S-FIL) method, is able to directly harvest nanogels from a silicon wafer substrate into

Fig. 1. Nanogel preparation by emulsion polymerization: a) with emulsion, b) without emulsion.

Fig. 2. Preparation of nanogels by cross-linking of amphiphilic block copo-lymer at core or shell of the polymer micelles in water.



aqueous buffers using a simple and biocompatible process (Figure 4).26 The method does not require particularly severe preparation conditions such as high temperature, high shear, organic solvents, or light exposure, allowing for effi cient encap-sulation of bioactive compounds. Using this method, peptide cross-linked PEG nanogels, in which antibodies or nucleic acids are encapsulated, have been synthesized. Particle shape and surface chemistry can be fi nely controlled with this method, which is diffi cult using other bottom-up methods.

3. Physically Cross-Linked Nanogels

3.1 Self-Assembled Nanogels Formed by Amphiphilic-Associating Polymers

Physically cross-linked nanogels can be prepared using non-covalent interactions between polymer chains, such as hydrogen bonds, Van der Waals forces, and electrostatic and hydrophobic interactions. It is generally diffi cult to obtain stable physically cross-linked nanogels with controlled sizes using such associating polymers because of their relatively weak non-covalent interactions. We have developed a new self-assembling method for the preparation of physically cross-linked nanogels using the controlled association of hydropho-bically modifi ed polymers in dilute aqueous solution.3 In particular, cholesterol-modifi ed polysaccharides have been

used to form stable monodispersive nanogels with a diameter of about 30 nm in water. The association of cholesteryl groups provides cross-linking points through hydrophobic interactions.

Generally, conventional self-assembled amphiphilic nanoparticles such as polysoaps (20–100 mol% hydrophobic group content) and proteins (ca. 50 mol% hydrophobic amino acid content) contain a considerable number of hydrophobic groups in the polymer chains. In contrast, hydrophobically modifi ed polymers have only small numbers (5 mol% or less) of hydrophobic groups. This means that they are not able to form core-shell type rigid nanoparticles, which have densely packed cores. Thus, the hydrophobically modifi ed polymers preferably form a hydrogel-like structure cross-linked by a large number of hydrophobic domains consisting of choles-teryl groups.

This self-assembly method, using hydrophobically modifi ed polymers, has proven to be an effi cient and versatile technique for preparing functional nanogels (Figure 5). Various nanogels have been obtained by the self-assembly of cholesterol-bearing poly(amino acids),27 cholesterol-bearing mannan,28 deoxycholic acid-bearing chitosan,29 bile acid-bear-ing dextran,30 synthetic polyelectrolytes with hydrophobic groups,31 and alkyl group-modifi ed poly(N-isopropylacryl-amide)–cholesterol-modifi ed polysaccharides mixture.18 Physi-cally cross-linked nanogels have advantages with respect to their biomedical applications, because toxic cross-linkers,

Fig. 3. Nanogel preparation by nanotemplate methods: a) liposome, b) gold nanoparticle.




catalysts, and byproducts are not necessary in the preparation process.

3.2 Self-Assembled Nanogels formed by Various Associating Polymers

Various intermolecular forces other than hydrophobic interac-tions have been utilized as a driving force in the preparation of physically cross-linking nanogels. For instance, a nanogel of water-soluble chitosan–poly(l-aspartic acid)–polyethylene glycol was prepared under relatively mild conditions using the complexation between polyelectrolytes through electrostatic interactions.32 Host-guest interactions with cyclodextrin have also been utilized to prepare nanogels using the association between cyclodextrin-bearing polymers and hydrophobic group-bearing polymers.33 Photoresponsive nanogels based on a spiropyrane-modifi ed pullulan34 and nanogels comprised of ethylenediaminetetraacetic acid-bearing chitosan capable of changing surface charge states in response to pH have been reported.35 Nanogels utilizing a wide range of intermolecular forces show great potential for preparation of novel stimulus-responsive nanogels.

By grafting hydrophobic polymer chains (polylactic acids) onto polysaccharides in place of low molecular weight hydro-phobic groups, more stable nanogels with relatively large hydrophobic domains have been obtained.36 Thermo-respon-sible materials that exhibit lower critical solution temperatures (LCSTs) in aqueous media have received particular atten-tion.37 For instance, the preparation of nanogels that enable heat-induced association and dissociation of polysaccharides partially grafted with short poly(N-isopropylacrylamide) (PNIPAM) chains have been reported.38 These polymers readily dissolve in water at room temperature. Above a

Fig. 4. Schematic representation of the step and fl ash imprint lithography (S-FIL) method.

Fig. 5. Nanogels formed with associating polymers and the AFM image.



lower critical solution temperature (LCST), PNIPAM-g-polysaccharides form nanogels that are physically cross-linked by the hydrophobic nanodomains generated by dehydration of PNIPAM (Figure 6). Biocompatible heat-induced nanogels have also been obtained by grafting poly(2-isopropyl-2-oxazoline) (PIPOZ),39 which is biocompatible, crystalline, and thermoresponsive, onto a polysaccharide. The assembly of the polymers is driven by the crystallization of PIPOZ chains, following heat-induced dehydration of the chains in water heated above the LCST.

3.3 Functions of Self-Assembled Amphiphilic Nanogels

3.3.1. Complexation with Proteins

One of the most notable characteristics of hydrophobically modifi ed polysaccharide nanogels is their dynamic property, which enables them to spontaneously trap proteins into the nanoscale hydrogel matrix. For instance, the nanogels of cho-lesteryl group-bearing pullulan (CHP) selectively interact with various proteins, primarily through hydrophobic interac-tions.40,41 The nanogels of the complex showed an excellent colloidal stability without any precipitation. One CHP nanogel complexed with approximately one bovine serum albumin (Mw 66,000), two α-chymotrypsin (Mw 25000), two myo-globin (Mw 17,800), four molecules of cytochrome c (Mw 12500), and fi ve molecules of insulin (Mw 5735). The maximum amount of protein complexed by CHP nanogels depends on the molecular weight (or size) and hydrophobicity of the protein. This is an interesting example of a host-guest interaction in a macromolecular system.

Most native enzymes were complexed with CHP nanogels at room temperature or physiological temperatures, and lost their enzyme activity in the complex.42 However, lipase was complexed in its native form and retained its activity even in the complex. In both cases, the complexed proteins gained substantial thermal stabilization owing to fi xation of the pro-teins or enzymes in the hydrogel matrix of nanogels.43

3.3.2 Chaperone-like Function of Self-Assembled Nanogels

In living systems, molecular chaperones selectively trap heat-denatured proteins or their intermediates, primarily by hydrophobic interactions, to prevent irreversible aggrega-tion owing to macromolecular host (molecular chaperone)-guest (protein) interactions.44 Then, with the aid of ATP and another co-chaperone, the host chaperone releases the protein in its refolded form. The molecular chaperone systems inspired us to explore new concepts in designing artifi cial molecular chaperones to assist protein folding. Designing an artifi cial host with a nanocage to bind denatured proteins and controlling the dynamics of catching and releasing proteins are indispensable for simulating the function of molecular chaperones.

We reported that amphiphilic nanogels, such as the CHP nanogels, act as artifi cial molecular chaperones. CHP nanogels trap heat denatured proteins45 or chemically denatured pro-teins (intermediates in the process of protein refolding after chemical denaturation by urea or guanidium hydrochlo-ride).46,47 Irreversible aggregation of denatured proteins was suppressed by complexation with nanogels. As a result, the colloidal and thermal stability of proteins signifi cantly increased. This is similar to the function of real molecular chaperone systems (Figure 7).

The protein release and refolding, which are induced by ATP in molecular chaperone systems, can also be simulated by the use of cyclodextrin as a nanogel chaperone system. CHP nanogels are dissociated upon complexation with β-CD, yield-ing CHP-CD complexes.48,49 The main driving force of the formation of nanogels is the association of the hydrophobic cholesteryl groups of CHP in water. Cyclodextrins are able to solubilize hydrophobic compounds in water by incorporation of the cyclodextrin into hydrophobic cavities. The CHP nano-gels dissociate upon complexation with β-CD to yield a dis-sociated CHP-CD complex, because the cholesteryl group is a suitable guest for β-cyclodextrin (β-CD).50 Thus, the dissocia-tion of the CHP nanogel–protein complex subsequently allows the release and renaturation of proteins.45

Fig. 6. Nanogels enable heat-induced association and dissociation of polysaccharides partially grafted with short poly(N-isopropylacrylamide) (PNIPAM) chains.




The chaperone-like activity of the CHP nanogel for refolding of acid-denatured green fl uorescent protein is com-parable to that of the native chaperone, GroEL.51 This nanogel system was also effective for renaturation of the inclusion body of a recombinant protein belonging to the serine protease family.46 CHP nanogels interact with monomers and oligo-mers of amyloid β-protein (Aβ) and the resulting complexes signifi cantly reduce Aβ toxicity in both primary cortical cultures and microglial cell cultures (Figure 8).52,53

3.3.3 Application to Drug Delivery Systems (DDS)

The molecular chaperone function is an important concept that has led to breakthroughs in DDS development, especially for protein or peptide delivery.54–58 Several studies have reported that complexes of nanogels with cancer antigen pro-teins can trigger an unprecedented immune response, making them useful in cancer immunotherapy.59–62 Clinical trials of a nanogel-cancer antigen protein complex as multifunctional cancer vaccines (activation of both CD8+ Killer T cells and CD4+ helper T cells) were initiated from 2004. The hydro-phobic antigen proteins were effectively complexed by a CHP nanogel without aggregation. The complexes (<50 nm) obtained were colloidally stable and effectively internalized antigen-presenting cells, such as dendritic cells, in vivo. Cat-ionic CHP nanogels have been found to be effective for anti-genic protein delivery for adjuvant-free intranasal vaccines,63 and for small interfering RNA (siRNA) delivery.64 They have also been reported to be effective as an artifi cial nucleic acid chaperone for oligonucleotides.65

4. Organic-Inorganic Hybrid Nanogels and Biomedical Application

Nanogels provide suitable reaction sites for mineralization owing to their high water content of the internal polymer

Fig. 7. Comparison of the mechanisms of natural (a) and artifi cial (b) molecular chaperones.

Fig. 8. Nanogels interact with monomers and oligomers of amyloid β-protein (Aβ) to reduce the Aβ toxicity by preventing aggregation of Aβ.

matrix and their large surface area. For example, nanoparticles of calcium phosphate and hydroxyapatite have been obtained using polysaccharide nanogels as scaffolds.66,67 The hybrid nanogels are useful as new intracellular protein carriers because of their excellent biocompatibility and biodegradability in addition to their appropriate mechanical stability.



Hybrid nanogels have been prepared by condensation of inorganic silanol groups (sol-gel reaction) grafted onto polysac-charides at an ambient temperature and pH without any cata-lysts or organic solvents (Figure 9).68 Hybrid nanogels can be rigidifi ed upon covalent cross-linking with inorganic siloxanes which enable them to provide additional platforms for func-tionalization with other silane coupling agents, titania, and hydroxyapatite. These stable hybrid nanogels are promising candidates for controlled-release DDS. The use of hybrid nanogels based on PNIPAM gels and tailored nanoporous silica has shown that the drug slowly diffuses out of the porous channels (i.e., by a nanodiffusion mechanism).69

A polyamine gel core of PEGylated nanogels was found to act as a nanoreactor and nanomatrix for the production and immobilization of gold nanoparticles through the self-reduction of aurate ions. The nanogel-gold hybrid functions as a smart nanoprobe for real-time monitoring of cancer therapy.70 Single gold nanorods encapsulated in PNIPAM nanogel71 have also been developed. These nanorods demon-strate photothermal phase transition and accumulation in local targeted sites irradiated by a near-infrared laser. The develop-ment of these PNIPAM-coated nanorods enables biomedical applications including targeted delivery, photothermal therapy, and bioimaging.

Monodisperse hybrid nanoparticles of quantum dots (QDs) have been developed by the simple mixing with

nanogels modifi ed with amino groups. Cationic nanogels have been utilized as novel carriers of the QDs for intra-cellular labeling (Figure 10).72,73 Hybrid nanoparticles can be effectively internalized into various human cells. The effi ciency of cellular uptake was found to be much higher than that of a conventional carrier, cationic liposome. Hybrid nanogels have a high potential for use in long-term live cell imaging.

Furthermore, various organic-inorganic hybrid nanogels, polysaccharide-magnetic nanogels for potential biomedical applications such as magnetic bioseparation, magnetic reso-nance imaging contrast agents, and hyperthermia treatments of cancer,74,75 hybrid nanogels consisting of lanthanum/euro-pium nanoparticles with unique optic properties and polyeth-ylene glycol,76 and fl uorescent nanogels involving arsenic sulfi de nanoclusters77 are all currently being developed, with the goal of applying them in nanomedicine.

Fig. 9. Hybrid nanogels prepared by condensation of inorganic silanol group (sol-gel reaction) grafted onto polysaccharide (a) and the TEM image (b) and size distribution (c).

Fig. 10. Hybrid nanogels as a novel carrier of quantum dots (QDs) for intracellular labeling.




5. Nanogel-Integrated Hydrogel

Conventional hydrogels have been widely used as functional materials in biotechnological and biomedical applications. However, designing hydrogels with a well-controlled nanodo-main structure remains challenging. We found that a nanogel-accumulated hydrogel could be formed when self-assembled CHP nanogels were dispersed in relatively high concentration (30 mg/mL or more; Figure 11a).78 To further develop nanogel bottom-up engineering, polymerizable nanogels can be employed as building blocks to control nanostructure in the macrogel. For this purpose, CHP nanogels containing meth-acryloyl groups as polymerizable units were designed (Fig. 11b).79 Novel nanogel cross-linked macrogels with immobi-lized artifi cial molecular chaperone functions80 and quick thermoresponsivity81 have been produced by polymerizing methacryloyl-bearing CHP (CHPMA) nanogels with various water-soluble monomers. Macrogels consisting of network structures of nanogels connected by biocompatible polymers can be formed by polymerization between nanogels and 2-methacryloyloxyethylphosphocholine in relatively high con-centration of the aqueous solution.

Another nanogel cross-linked macrogel has been devel-oped by Michael addition of CHPMA nanogels with multi-branched PEGs terminated with thiol groups. The linkages between nanogels and PEGs in this system are degradable so that the nanogels as building units are released as the gel dis-solves. Such degradable nanogel cross-linked hydrogels are

useful as a novel scaffold material for controlled release in regenerative medicine, including bone regeneration.82

Nanogel cross-linked nanoparticles have also been devel-oped. In these nanoparticles, the reaction for the nanogel cross-linking is performed under relatively dilute conditions. In this system, physically cross-linked nanogels can be stabilized by chemical cross-linking. Thus, these nanoparticles can be utilized as injectable carriers capable of controlled release of proteins such as cytokines over relatively long periods. For example, a raspberry-like assembly of nanogels was prepared from the acrylate-modifi ed CHP nanogel and the PEGSH (Fig. 11c).83 Interleukin 12 (IL-12), an immunostimulatory cytokine, can be encapsulated in the nanogel cross-linked nanoparticle and released over a prolonged period of time both in vitro and in vivo. The bottom-up nanogel fabrication method can thus be used to create tailor-made functional hydrogel materials.

6. Conclusions

Biomedical applications of nanogels have made rapid progress in the last 10 years. These developments have allowed control-ling over properties of nanogels, such as size, stability, surface functionality for bioconjugation, and biodegradability, which has enabled an expansion in the range of drugs and pharma-cokinetic profi les available in DDS applications. A current challenge is the development of strategies for the delivery of more fragile drugs such as proteins, antibodies, or nucleic acids, which are easily unfolded or inactivated under physio-logical conditions. To address this issue, the chaperone-like function of the nanogels presented in this article is an impor-tant concept that can lead to breakthroughs in the effective delivery of these unstable drugs.

One future goal of nanogel research should be the use of these smart nanogels as individual components for building an integrated nanosystem using nanogel engineering. This nanogel engineering provides a new paradigm for development of nanogel-based biomaterial with well-organized three-dimen-sional structure, multiple functions, sensitivity to a range of different stimuli, and programmed responses that can be con-trolled temporally and spatially. Such hierarchically integrated nanosystems, making use of nanogel properties, will provide a material superior to individual nanogel components.

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