Nanogels with Selective Intracellular Reactivity for Intracellular Tracking and Delivery

Abstract A multimodal approach for hydrogel‐based nanoparticles was developed to selectively allow molecular conjugated species to either be released inside the cell or remain connected to the polymer network. Using the intrinsic difference in reactivity between esters and amides, nanogels with an amide‐conjugated dye could be tracked intracellularly localizing next to the nucleus, while ester‐conjugation allowed for liberation of the molecular species from the hydrogel network inside the cell, enabling delivery throughout the cytoplasm. The release was a result of particle exposure to the intracellular environment. The conjugation approach and polymer network building rely on the same chemistry and provide a diverse range of possibilities to be used in nanomedicine and theranostic approaches.

Abstract: Am ultimodal approach for hydrogel-based nanoparticles was developed to selectively allow molecular conjugated species to either be released inside the cell or remain connected to the polymer network. Using the intrinsic differencei nr eactivity between esters and amides, nanogels with an amide-conjugated dye could be trackedintracellularlyl ocalizingnext to the nucleus, while ester-conjugation allowed for liberation of the molecular speciesf rom the hydrogeln etwork inside the cell, enabling delivery throughout the cytoplasm. The releasewas ar esult of particlee xposure to the intracellular environment. The conjugationa pproach and polymer network buildingr ely on the same chemistry and provide ad iverse range of possibilities to be used in nanomedicine and theranostic approaches.
Within the fieldo fn anomedicine, delivery vehicles are regarded as one of the major strategies to successfully protect and deliver pharmaceutical components. [1] However,i ta lso becomes more apparent that different delivery strategies are neededf or different diseases and different cell types. [2] In drug delivery,b oth the release mechanism and the body distribution of the vehicle play an important role. Theranostic approaches, which combine diagnosis and treatment of disease, serve to visualize drug delivery vehicles. [2][3][4] So far,m any of the theranostic approaches are performed with solid nanoparticles (inorganic/organic) or liposomes/polymersomes. [2][3][4][5] Am ajor advantage of liposomes and polymersomes is that these allow for the use of the completep article volume for drug loading, while for most of the solid particles only the particle surfacei s available. However,s olids tructures have generally an en-hanced stability towards different (bio)chemical environments, solvents, and mechanical stress.
Hydrogel nanoparticles fulfill an importantn iche by combining high drug loadingc apacity with tunable stabilitya nd resilience.T hey consist of ac ovalent polymer network which provides the stability to cope with chemical environments and solvent compositions, andc an be designed to respond to various stimuli,s uch as temperature,p H, and light. [6] On top of that, hydrogel nanoparticles can undergoh igh deformationsw ithout breaking. [7] Hydrogel nanoparticles or nanogels, have been used in various fields of research includingc atalysis, [8,9] selectived iagnostics and delivery, [10,11] and anti-fouling coatings. [12,13] Even though these nanogelshave found their wayinto the biomedical field, [14,15] theranostics and multimodalities remainu nderdeveloped and mostly hybrid structuresa re being used (inorganic/organic). [16][17][18] Few examples have been developedt hat rely solely on hydrogel structures [17,[19][20][21][22][23] and when doing so, the multimodality originates from as ingle molecular species, [24] whichl imits the general applicability. Therefore, creating a more generalized approach based on the same chemistry but with the possibility of easy exchanging the actives tructure provides ap owerful approacha nd furtherd evelop the field of multimodaln anogels in nanomedicine.
Our aim was to utilize the intrinsic difference in reactivity betweene ster-and amide-conjugationto tune the susceptibility of nanogel hydrolysis towards the intracellular environment and thereby controlling selective release of incorporated moleculars tructures.E sterases, found in the intracellular environment,a re known to have chemical selectivity. [25] The modalities with differenti ntracellulars tability were incorporatedw ithout altering the overall development of the nanogel andu sing acrylice sters,r elease of incorporated molecules was triggered withint he cell whileo utside the cell released id not occur. Using acrylamide conjugation,r eleasew as absent both outside and inside the cell, and particlet racking was facilitated. Intracellular stabilityo ft he nanogels was corroborated by the response of the nanogels toward cell lysate, which triggered similar releasep rofiles. The presented versatile and innovative approachw ill trigger developmento fn ew multimodal particles for drug delivery and theranostics and exemplifies easy diversification using simplec hemical approaches and introducing these within the same particle without affecting their function.
The nanogel formation is based on well-established precipitation polymerization (Scheme1). [25] The initiation of afree radical polymerization leads to small oligomers that have different solubility than the used monomersu nder set conditions, which leads to the precipitation and formation of small colloids. These colloids will grow in size and after termination of the polymerization reactiona nd reestablishmento fs olubilizing conditions of the polymers, the network within the colloids will be rehydrated, resulting in an anogel.B yu sing am ixture of monomers, various functionalities can be introduced such as degradability,r esponsiveness (temperature, pH, light, redox reactions), simplyb ya dding other monomers that will take part in the polymerization process. [25] Here, fluorescein-acrylate (FL-Ac) and Nile blue acrylamide (NB-AAm) have been used to demonstrate the principle of using the specific intracellular environmentt os electively releaseo rr etain components in the nanogel, respectively ( Figure S1). N-Isopropylmethacrylamide (NIPMAM)i su sed as the responsive unit that facilitates the use of precipitation polymerization. The volumep hase transition temperature (VPTT) of NIPMAM is around 45 8Ca nd above this temperature, dehydration occurs followed by precipitation and colloid formation.This process can be repeated, which will provide as hell with ad ifferent composition around the initially formed core particle. [26,27] After the formation of the FL and NB-labeled core particles, as hell consisting of NIPMAM, BIS, and 3-acrylamidophenylboronic acid (APBA)w as formed (Scheme 1) which was evaluated by 1 HNMR spectroscopy (Figure S2). It is know that APBA is enhancing cellular uptake in cancer cells, due to its ability to bind to the overexpressed sialic acid on the surfaceo fc ancer cells. [28][29][30] The differencei n extracellular and intracellular reactivity as well as the intrinsic stabilityd ifference between esters and amides facilitatecontrol over the release of incorporated molecules from the nanogel at the desired location (intracellular). After uptake of nanogels by cells via endocytosis, it was envisioned that there would be af ast releaseo fe ster-conjugated structures from the nanogel due to the presence of esterases in endosomes,w hereas the amide-conjugated structures will remaina ssociated with the nanogel as illustrated in Scheme 1a nd Figure S1.
The nanogelsu sed are primarily based on NIPMAM, which will dehydrate upon increasing the temperature to above the VPTT of 45 8C, which induces ad ecrease in size as shown by the temperature dependent dynamic light scattering (DLS) measurements (FigureS3A). The VPTT is far above 37 8C, that is, body temperature, at which cell cultures are primarily done. Clearly, similar handling and incubation temperatures will apply in ac linicals etting. At these conditions, temperature-induced changes in the nanogels are not desired, as our aim is to elucidate the releasep rofiles of the nanogelsi nr esponse to the intracellular environment. Additionally,acollapsed matrix of the nanogel would inhibitp ropere xposure of the FL-Ac nanogel to the intracellular esterase and could inhibit the hydrolysis. At room temperature (20 8C) the hydrodynamic diameter for NB-AAm nanogel was 460 nm while at 37 8Ct he diameter reduced slightly to 430 nm. For the FL-Ac nanogel the difference in hydrodynamic diameter was negligible, both around 300 nm (Figure S3 A). The diameter of both nanogels decreased upon increasing the temperature far above 37 8Ca nd is therefore not considered to have any influence on cellular uptakeo rr elease events. The transmission electron microscopy (TEM) images confirm the presence of particle structures (Figure S3 B,C). The diameters were approximately 320 nm for the NB-AAm nanogel and2 00 nm for FL-Ac nanogel,w hich are slightly smaller than the hydrodynamic diameters in the swollen state observed by using DLS as the nanogels are in dry state when measured with TEM.
It was envisioned that the intrinsic difference in reactivity of ester-and amide-conjugated structures could be utilized to control the releasep rofile of nanogels following their exposure to the intracellulare nvironment. Intracellular esterases are capable of hydrolyzing av ariety of substrates, while amidases are more selective towards pecific peptide bonds. [31] After nanogel internalization by cells, the difference in enzymatic susceptibility is utilized to initiate ar elease of ester-conjugated structures, here af luorescein-ester conjugated moiety,w hereas amide-conjugated structures, here aN ile blue-amide conjugated moiety,r emain confined to the nanogel.
Confocal laser scanning microscopy (CLSM) of MCF-7 breast cancer cells incubated with NB-AAm nanogel for 2hrevealed a punctatef luorescentp attern in the cell cytosol, suggesting the presence of nanogelsi ne ndosomes ( Figure 1A). Incubation of MCF-7 cells with FL-Ac nanogel showedi na ddition to fluorescent spots, ad iffuse cytosolic fluorescence. In addition, reticular structures werev isible next to the nucleus, reminiscent of endoplasmic reticulum ( Figure 1A). Clearly,t he two types of nanogelsd isplayed distinct intracellulard istributions following their incubation with MCF-7 cells. To exclude that the intracel-Scheme1.Synthetic approach for the multimodal nanogels through controlled precipitation polymerization in at wo-stepf ashion to create the core and subsequently the cellrecognizing shell.Below,aschematic representation of the overallbehavior of release and trackingo utside and within the cell. lular distribution differences were ar esulto fd ifferencesi n nanogel cytotoxicity,acell viabilitya ssay was performed. Both types of nanogels were identified to be non-toxic at the concentrations used within the experiments ( Figure S4). The internalization of the nanogels rather than being confined to the surfaceo ft he cells was confirmed analyzing the fluorescence signal in the z-direction ( Figure S5), which shows that the fluorescent signal is present within the cells.
Next, MCF-7 cellsw erei ncubated with am ixture of NB-AAm and FL-Ac nanogels. Figure 1B shows that both nanogels were efficiently internalized by the cells, to as imilare xtent as upon separatei ncubations with the nanogels. Furthermore,M CF-7 cells showedapartial overlapo ff luoresceins pots with Nile blue punctate in the cell cytosol, which mostl ikely indicates colocalization of the fluorescent nanogelsw ithin endosomal structures ( Figure 1B and Figure S6). In addition, the fluorescein label was widely distributed over the entire cell. This highly diffuse pattern of the FL-Ac label indicates that the dye was efficientlyc leaved from the nanogel, most likely due to the presence of endosomal esterases, and released from endosomes.T ypically,e sterases have ab roader range of substrate acceptance for hydrolysis than amidases. Therefore, the NB-AAm label remained associatedw ith the nanogel and confined to endosomes. Herewith, we show as imple approach to trigger intracellular cargo release or enable cargo confinement, by means of an intrinsic difference in reactivity of build-inlinkages inside the nanogel network.
While the selectivity was illustrated using am ixture of the nanogels, for theranostic and/ord ual drug delivery approaches, both properties should be incorporated into the same nanogel without affecting the individual characteristics. Therefore, an anogel was prepared containing both the esterand amide-conjugated dyes and the same intracellular release study was performed, which also excludes the possibility that the differences in fluorescencep atterns observed for the two types of nanogelsa re caused by differences in their size (Figure S3). Figure 2s hows the same distinct patterns for fluorescein and Nile blue as was observed from mixing the two nanogels, only now both originate from the same nanogel. The fluorescences ignal from the ester-conjugated fluoresceini s found both punctuated and cytosolic while the signal from the amide-conjugated Nile blue is exclusively punctuated. This conceptual approach illustrates the deliveryp otentialc ombined with traceability of such nanogels.
Uptake of nanoparticles by cells primarily occurs via endocytosis. If nanogels are internalizedv ia the endosomal pathway, then hydrolysis could also be facilitatedb yt he lowering in pH. It is well knownt hat the pH within an endosome decreases to pH value of 6.5f or early endosomes and continues to be low-   [32] This loweringo fp He nhances the hydrolysis of esters. In order to identify if FL-Ac releasef rom the nanogels is dependent on a drop in (endosomal) pH, we exposed the differentn anogels to cell lysate at pH % 7. The cell lysate contains all enzymes found intracellularly,w ithout the need of going through the uptake process. Thereby,t he pH shifts at the different endosomal stages are omitted. After exposure of the nanogelst oc ell lysate and subsequently removing the nanogels via ultracentrifugation ( Figure S7), the fluorescence signal of the supernatant was determined in order to analyze the release of fluorescent cargo from the nanogels. Fluorescencet hat was released from nanogelse xposed to water served as ac ontrol.F igure S7 shows the absence of fluorescences ignali nt he supernatant of NB-AAmn anogels exposed to cell lysate, which was identical to that of the nanogel not exposed to cell lysate. In sharp contrast, the FL-Ac nanogel supernatant did not show fluorescence signal after exposure to water (Figure 3), while in the presence of HBSS buffer as mall fluorescences ignal was detected.H owever,w hen in presence of cell lysate, the fluorescence intensitya tt he maximum wavelength (l max = 518 nm) is 13 times highera sc ompared to HBSS buffer alone, showing the substantial influence of the intracellular environment on the release. As the pH within these experiments was maintained at % 7, we can conclude that cell lysatec omponents, presumably esterases, are able to selectively hydrolyzet he FL-Ac conjugates. These data were supported by bafilomycinA1 inhibitione xperiments.M CF-7 cells were treated with nanogels in the absence and presence of bafilomycin A1, whichi nhibits the acidification of endosomes. As shown in Figure S8, the FL-Ac nanogel displayed the same releasep attern in the presence of bafilomycin A1 as in its absence, indicatingt hat the FL-Ac can be released from the nanogel and redistributedt hroughout the cell without exposure to low pH.
We show that the differencei ni ntracellularr eactivity between esters and amides providesapowerful tool to decide between intracellularc argo release and confinementi nn anogels. Controlled releasem ay serve to mediate drug delivery, while confined cargo,f or example, fluorescence, can be used for nanogel tracking. In the field of theranostics, it is pertinent that differentf unctions have tailored conjugation behavior. Tracer and drug releasei st hereby tunable and the approach opensu pn ew possibilities for designing multimodalh ydrogel nanoparticles for medical applications.M ore diverse chemistries are still to be explored, but it has already been shown that tuning the shape (linear vs. branched) and hydrophobicity of aliphatic esters resulted in differencesi nr elease rate in solid acrylate-based nanoparticles. [33] By diversifying the ester-conjugation in terms of hydrophobicity and steric hindrance, sequentialr elease approaches would be possible from the same particlew hile maintaining the detectionc apabilities of using the amide-conjugation. In short, hydrogel nanoparticles, socalled nanogels, find their way into biomedical applications because of the possibility of loading the entire particlev olume with (therapeutica nd/ord iagnostic) substances [21] ,t he diverse range of chemical components that can be included, as well as the ease of formation and upscaling. Introducing the multimodal approach as depicted here, these particles will become even more widelya pplicable.