Cross-Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery

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Cross Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery: Application to Polymeric Nanoparticles Marianna Galliani, Melissa Santi, Ambra Del Grosso, Antonella Cecchettini, Filippo M Santorelli, Sandra L Hofmann, Jui-Yun Lu, Lucia Angella, Marco Cecchini, and Giovanni Signore Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00206 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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Bioconjugate Chemistry

Cross Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery: Application to Polymeric Nanoparticles Marianna Galliani1,2 *, Melissa Santi1,2, Ambra Del Grosso2, Antonella Cecchettini3,4, Filippo Maria Santorelli5, Sandra L Hofmann6, Jui-Yun Lu6, Lucia Angella2, Marco Cecchini2 and Giovanni Signore1,2* 1

Center of Nanotechnology Innovation@NEST, Istituto Italiano di Tecnologia, 56127 Pisa, Italy NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, 56127 Pisa, Italy Institute of Clinical Physiology-CNR, 56127 Pisa, Italy 4 Dept. of Experimental and Clinical Medicine, University of Pisa, 56127 Pisa, Italy 5 Molecular Medicine, IRCCS Stella Maris, 56128 Pisa, Italy 6 University of Texas Southwestern Medical Center, Dallas, 75390 TX, US 2 3

* Corresponding Author E-mail address: [email protected] ; [email protected]

KEYWORDS protein delivery • enzyme replacement therapy • PLGA nanoparticles ABSTRACT Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross linked enzyme aggregates (CLEAs) encapsulation into poly(lactide-co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease) and beta glucosidase (bGLU; defective in Gaucher’s disease). We demonstrated that our system allows encapsulating enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEAs NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48h. Lastly, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEAs NPs.

INTRODUCTION Protein delivery represents to date one of the most promising strategies for the treatment of diseases such as cancers, diabetes and rare diseases like lysosomal storage disorders (LSDs)1. Unfortunately, systemic administration of proteins and large molecules suffers from several limitations, including degradation, poor biodistribution and inability to cross most biological barriers. This heavily impairs the efficacy of protein and enzyme replacement therapies for many diseases, in particular for those involving the central nervous system, as the LSDs2. Nanoparticle (NP)-based carriers represent a promising platform for protein and enzyme delivery, as they can protect the payload from degradation and target specific areas even beyond the blood brain barrier (BBB)3,4. Thus, preparation of stable, efficient formulations of active proteins in nanostructured vectors is an actively researched topic. Liposomes5–7 and polymeric NPs8–11 have been extensively investigated, usually with heterogeneous results due to unpredictable encapsulation efficiency and activity retention. Among the investigated materials for NP synthesis, poly (lactide-co-glycolide) (PLGA) has received special attention, being a biocompatible, nontoxic and FDA approved polymer for intravenous administration in humans12. However, successful encapsulation of proteins and enzymes in PLGA NPs is still an open issue. Most studies on protein loading in PLGA NPs are based on double emulsion/solvent evaporation method13–15 that

requires harsh conditions (such as halogenated solvents and sonication) which could easily denature proteins and enzymes. Several efforts have been made to adapt other methods, such as nanoprecipitation, to protein loading16–18, whereas recent works investigated the use of either polycation-modified PLGA19,20 or copolymers of the analogous poly (lactic acid) and poly (ethylene glycol) (PLA-PEG)21. Although some of these strategies led to fairly good results, to date there is no reliable approach for efficient loading of enzymes with good encapsulation efficiency and activity retention. In the framework of our interest in drug delivery in LSDs22,23, we here demonstrate a new encapsulation strategy that allows loading enzymes with excellent efficiency and activity retention. RESULTS AND DISCUSSION We investigated the use of cross linked enzyme aggregates (CLEAs). CLEAs were firstly described in the early 2000s, and are produced by precipitation of the enzyme into a water-miscible solvent together with crosslinking of the physical aggregates. CLEAs are usually stable in organic solvents and show unaltered and in some cases even enhanced - enzymatic activity compared with the parent enzymes24–27.

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Our approach to the synthesis of CLEAs-loaded NPs (CLEAs NPs) was based on a two-step process. In the first step, CLEAs were synthesized by adding the enzyme solution to acetone in presence of glutaraldehyde, resulting in Schiff base formation and subsequent crosslinking of the precipitated enzyme (Figure 1a). In the second step, CLEAs NPs were produced by nanoprecipitation (Figure 1b). CLEAs and PLGA were mixed in acetone and added dropwise to an aqueous solution of sodium cholate.

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between aqueous and organic phase of EAs, free enzyme and CLEAs, whose surface lysine residues are derivatized and made hydrophobic by the cross linker. This could lead to different water solubility, and hence lowered encapsulation efficiency for the more hydrophilic underivatized enzymes. EE% (SEM) Loaded enzyme PPT1 GALC aGLU bGLU

CLEAs NPs

EAs NPs

DE NPs

88 (11) 47 (7) 13.8 (0.3) 69 (6)

0.9 (0.4) 35 (3) 31 (3) n.d.

32 (7) 26 (3) 9 (3) 40 (11)

Table 1 Encapsulation efficiency (EE) of cross-linked enzyme aggregates (CLEAs NPs), enzyme aggregates (EAs NPs), and double emulsion (DE NPs) nanoparticles loaded with PPT1, GALC, aGLU or bGLU. n.d. = no data, the obtained value is equal to or lower than the blank value for all batches. SEM = standard error of the mean of three independent experiments.

Figure 1 Schematic illustration of (a) CLEAs and (b) CLEAs NPs synthesis.

When transferred into water, PLGA precipitates and spontaneously encapsulates CLEAs in NPs that can be collected by centrifugation. To better understand the role of the crosslinker, we also prepared control enzyme aggregates-loaded NPs (EAs NPs) by precipitating the enzyme in absence of crosslinker in the first step (leading to physical enzyme aggregates, EAs). Next, we evaluated physico-chemical properties and encapsulation efficiency of CLEAs NPs. CLEAs encapsulation affects neither the size nor the surface charge of NPs compared to empty NPs produced with the same strategy (Table S1). Indeed, all formulations show hydrodynamic diameter ranging from 130 to 200 nm and zeta potential around -35 mV, in keeping with values usually observed for PLGA NPs obtained by nanoprecipitation. Enzyme encapsulation efficiency in NPs was determined via ninhydrin assay after amino acid digestion, since other approaches based on the quantification of intact protein complexes afforded not reproducible results. In all cases encapsulation efficiency for CLEAs NPs largely exceeds what found for double emulsion NPs (DE NPs) and is far superior to what observed for EAs in all cases except for aGLU (Table 1). This behaviour is easily rationalizable considering a different partitioning equilibrium

Next, we examined more in detail whether CLEAs are actually encapsulated in the inner part of the NPs or rather adhered to the surface. To this end, we labelled CLEAs with Atto488 (488-CLEAs) and encapsulated them into NPs tagged with Atto633-labelled PLGA (633-NPs). The internalization of 488-CLEAs into 633NPs was first verified by confocal imaging and colocalization analysis. Confocal images of 488CLEAs 633-NPs (Figure S2) revealed that all green labelled CLEAs are associated with red labelled NPs (Manders’ coefficient: >0.7 in all formulations), while several empty 633-NPs are also present in the samples (Manders’ coefficient ≤ 0.5 (Figure 2a)). To confirm this result, we performed a KI quenching assay, which provides an evaluation of the interaction between a fluorophore and the water environment by exploiting the well-known fluorescence quenching effect of a KI solution. Stern-Volmer plot of 488CLEAs quenching shows that for all formulations, encapsulated 488-CLEAs have a lower Stern-Volmer quenching constant (KSV) respect to 488-CLEAs outside NPs, reflected by a lower slope of the regression line (Figure 2b and Table S2). This suggests that CLEAs are encapsulated in the inner part of the NP and effectively shielded from the solvent. Remarkably, the ability of NPs to decrease the quenching rate of embedded compounds is also reflected by the low Ksv of 633-PLGA, that is screened by the NP itself (Figure S3 and Table S3). We then evaluated the release profile of PPT1 488CLEAs 633-NPs in neutral and acidic environment at 37°C (Figure 3a). In both cases PPT1 release profiles are biphasic and typical of PLGA NPs, characterized by an initial burst release followed by a sustained release11,28. Release profile is in line with what previously reported on PLGA nanoparticles in neutral and acidic media. The different behaviour could be

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related to the predominance of PLGA erosion or acidcatalysed hydrolysis at pH=7.4 and 4.5, respectively29,30. The Stern-Volmer plot evidences that virtually no enzyme is present on the surface of the NP, thus the observed burst release is likely due to the release of a fraction of enzyme located in proximity to the surface. We then evaluated the loss of activity due to the process of crosslinking and encapsulation in NPs (Table 2). As expected, enzymatic activity of EAs is higher than in CLEAs, due to the structural modifications occurring to the protein upon crosslinking.

could be rationalized by taking into account the wellknown enhancement of catalytic activity of NPimmobilized enzymes, which in our case could apply to the immobilized CLEAs31,32. Furthermore, the mildly acidic environment typical of PLGA33 might promote hydrolysis of imine bonds in the crosslinked CLEAs, thus restoring pristine activity. To verify this hypothesis, we prepared a batch of reduced bGLU CLEAs (R-CLEAs), where the Schiff bases formed upon crosslinking were converted into irreversible amine bonds via reductive amination34 and encapsulated them in PLGA NPs (R-CLEAs NPs). We then evaluated activity recovery upon encapsulation compared with the precursor R-CLEAs. R-CLEAs show only 1.5-fold activity increase upon encapsulation in NPs, compared with the 2.3-fold increase shown by CLEAs (Figure S5), thus evidencing that the presence of a reversible crosslinking between enzymes is a fundamental requirement for reestablishment of catalytic activity. The slight activity increase observed in R-CLEAs NPs can be due to the incomplete reduction of the imine bonds in a structurally complex matrix such as a protein aggregate. AY% (SEM) Loaded CLEAs CLEAs enzyme NPs PPT1 GALC aGLU bGLU

6 (2) 64 (11) 28 (4) 59 (6) n.d. n.d. 25 (10) 61 (13)

EAs

EAs NPs

DE NPs

54 (2) 27 (2) 35 (4) 52 (7)

27 (6) 16 (2) 37 (3) n.d.

0.5 (0.2) n.d. n.d. 0.1 (0.1)

Table 2 Activity yield (AY) of CLEAs, CLEAs NPs, EAs, EAs NPs and DE NPs loaded with PPT1, GALC, aGLU or bGLU. n.d. = no data, the obtained value is equal to or lower than the blank value for all batches. SEM = standard error of the mean of three independent experiments.

Figure 2 (a) Manders’ coefficient resulting from colocalization analysis of 488-CLEAs 633-NPs formulated with PPT1, GALC, aGLU and bGLU. Error bars represent the standard error of the mean of 10 analysis. (b) Stern Volmer plot resulting from quenching of 488-CLEAs in 488CLEAs 633-NPs (green points) and 488-CLEAs outside 633NPs (red points) with increasing concentrations of KI. F0 and F are the fluorescence intensities in absence and in presence of the quencher, respectively.

This pattern is completely reversed when loaded NPs are considered. EAs usually experience a significant loss of activity upon encapsulation in PLGA, while CLEAs loaded in NPs show increased activity compared with CLEAs precursor, except for aGLU, which remains completely inactive. The apparently counterintuitive activity increase upon encapsulation

aGLU is the sole enzyme, among those tested, that is not suitable for CLEAs encapsulation, since it completely loses activity after CLEAs formation. Notably, activity is not restored after encapsulation in PLGA. Since no lysine residue is present at the catalytic site of aGLU35, it is tempting to assume that irreversible conformational changes take place in proximity of the catalytic site as a result of the crosslinking process. Note that enzymes loaded with the double emulsion method, despite a reasonably high encapsulation efficiency (usually between 25% and 42%, Table 1) show a nearly complete loss of activity (Table 2), thus confirming that double emulsion is not suitable for efficient encapsulation with maintained activity. Finally, we evaluated in vitro behaviour of enzymeloaded NPs. Preliminary to in vitro experiments, we evaluated cytotoxicity of PPT1 CLEAs NPs (see Figure S6), and we did not observe any toxic effect. First, we evaluated the release profile using

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fluorescently labelled PPT1 488-CLEAs 633-NPs in NIH-3T3 cells by means of confocal microscopy at different time points (Figure 3b and S7). Imaging 1 hour upon treatment shows extensive colocalization of CLEAs and NPs in discrete aggregates. After 6 hours, CLEAs and NPs are still colocalized but distributed as vesicles inside cells, indicating compartmentalization at lysosomal level. After 24 hours red labelled PLGA is present at vesicular level in most cells, while cells showing only green vesiculated PPT1 are also present. This suggests that PPT1 is being released and follows the expected trafficking and secretion pathway typical of PPT136. After 48 hours only few cells show red vesicles, whereas most cells show green vesicles due to released and trafficked PPT1. Red fluorescence is mainly concentrated in big vesicles or aggregates either within or outside cells, suggesting that NPs are processed and exocytosed as expected37. These results are also confirmed by a more quantitative colocalization analysis (Figure S8) that shows increasing red/green and decreasing green/red colocalization over time, in agreement with the spreading of enzyme and concomitant compartmentalization of PLGA residues. Notably, incubation with free enzyme highlights extensive endocytosis in short times, leading to a highly vesiculated signal. Colocalization with empty nanoparticles co-administered to the cells evidences that common fate of both species is in the lysosomal compartment after 6-24 hours. However, the enzyme mostly disappears after 24-48 hours, presumably owing to constitutive cell recycling (see Figure S9). Since this disappearance kinetics is markedly different from what observed using PPT1 CLEAs NPs, it is tempting to assume that NPs are able to control the release and prolongate the half-life of the enzyme inside the cell.

Figure 3 (a) Release profile of PPT1 488-CLEAs 633-NPs in acidic and neutral buffer at 37°C. (b) Confocal images of NIH-3T3 cells treated with PPT1 488-CLEAs 633-NPs 1, 6, 24 and 48 hours after incubation with NPs. Figures display red/green (NPs/CLEAs) superimposed images. Scale bars = 25 µm. Single green/red channel and bright field are reported in Figure S5.

Lastly, we set up a proof of concept experiment to evaluate the ability of PPT1 CLEAs NPs to restore enzymatic activity in vitro in a line of primary fibroblasts (cln1-pt9) harboring the homozygous R122W mutation in the PPT1 gene. To this end, we produced a targeted version of PLGA NPs using a mixture of plain and Tf2-derivatized PLGA. Peptide Tf2 was recently described38, and recognizes holotransferrin without altering its binding capability, thus allowing indirect targeting of transferrin receptor (TfR). Given the high density of TfR on the BBB, Tf2 decorated NPs could allow delivery to the central nervous system. The presence of Tf2 on the NP surface did not affect the NPs properties in terms of size, zeta potential, encapsulation efficiency and activity yield, apart from a minor decrease in encapsulation efficiency (Table S4). PPT1 CLEAs NPs and PPT1 CLEAs Tf2 NPs were administered to the cells and after incubation we assayed the cell enzymatic activity. As shown in Figure 4, both targeted and untargeted NPs promote recovery of enzymatic activity, although with different efficiency. At low doses (110 U) enzymatic recovery obtained using free PPT1 reaches plateau, likely reflecting saturation in the enzyme uptake mechanism. Note that enzymatic recovery is slightly higher for cells treated with Tf2-modified NPs respect to untargeted NPs, probably due to increased NPs uptake in presence of Tf2, which can activate receptor-mediated endocytosis.

Figure 4 Enzymatic activity of primary fibroblasts derived from NLC1 patients after treatment with increasing doses of PPT1, PPT1 CLEAs Tf2 NPs and PPT1 CLEAs NPs. unit 4


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(U) = amount of enzyme that catalyzes 1 nmol of substrate per hour. U/µg = Unit of enzyme per microgram of cell lysate. Error bars represent the standard error of the mean of 3 experiments.

CONCLUSION In summary, we developed a simple and general approach for the encapsulation of enzymes in PLGA NPs based on the formation of CLEAs, that is easily adaptable to different proteins and polymeric NPs. Enzymes are encapsulated with excellent efficiency and activity retention, taking a significant step forward compared to traditional synthesis methods such as double emulsion/solvent evaporation. We also demonstrated that enzyme-loaded NPs can be administered to primary NCL1 skin fibroblasts, fully restoring enzymatic activity and paving the way to the development of more effective enzyme replacement therapies. EXPERIMENTAL Materials Poly (D,L-lactide-co-glycolide) (PLGA; Resomer® RG 503H and Resomer® RG 752H) was purchased from SigmaAldrich and used as received. Recombinant human palmitoyl protein thioesterase 1 (PPT1) was produced according to a reported procedure39. Recombinant alpha-glucosidase (aGLU) from Bacillus stearothermophilus was purchased from Creative Enzymes (USA) and used as received. Beta-glucosidase (bGLU) from almonds was purchased from Sigma-Aldrich and used as received. Atto 488 NHS-ester and Atto 633 amine were purchased by Atto-TEC GmbH (Germany) and used as 10 mg/ml stock solutions in dimethylsulphoxide (DMSO). All other chemicals were purchased from Sigma-Aldrich unless otherwise specified. All chemicals were used as received. CLEAs synthesis 200 µl of 1 mg/ml enzyme solution in water were added dropwise to 600 µl of acetone simultaneously with 2 µl of 25% glutaraldehyde water solution under stirring in ice bath. The mixture was stirred overnight at 4°C then 200 µl of acetone were added. The suspension was centrifuged (13200 rpm, 4°C, 30 minutes) and washed twice with 1 ml of acetone. CLEAs were suspended in 1 ml of acetone and stored at -20°C until use. EAs were prepared according to the same procedure without addition of glutaraldehyde. CLEAs NPs synthesis Generally, 200 µl of CLEAs were added to 200 µl of a solution composed of PLGA 752H (8 mg) and PLGA 503H (2 mg) in acetone. The mixture was added dropwise to 1200 µl of 2% (w/v) sodium cholate in water under stirring. The resulting NPs suspension was centrifuged (13200 rpm, 4°C, 20 minutes) and washed twice with 500 µl of water. Obtained CLEAs NPs were suspended in 500 µl of 10 mg/ml trehalose aqueous solution and stored at -20°C until use. EAs NPs were produced following the same procedure by replacing CLEAs with EAs. TF2-modified NPs were produced by replacing PLGA 503H with PLGA-TF2 (see Supporting Information). Fluorescently labelled NPs (488CLEAs 633-NPs) were produced by replacing CLEAs with 488-CLEAs and by adding 0.1 mg of 633-PLGA to the

organic mixture (see Supporting Information). Empty NPs were prepared with the same protocol by replacing CLEAs with the same volume of acetone.

Encapsulation efficiency determination The amount of enzyme in the samples was determined via ninhydrin assay as described by Starcher40. Ninhydrin reagent was prepared as follows: 200 mg of ninhydrin were dissolved in 7.5 ml of ethylene glycol, then 2.5 ml of 4N acetate buffer (pH 5.5) and 250 µl of 100 mg/ml stannous chloride solution in ethylene glycol were added. Samples and standards were first digested by adding 50 µl of 12M HCl and 100 µl of 6M HCl to 50 µl of sample. The mixture was incubated at 95°C and then dried under vacuum. 50 µl of deionized water were added to the residue and 10 µl of this solution were added to 110 µl of ninhydrin reagent. The mixture was incubated at 95°C for 15 minutes then 50 µl were transferred in a 96-well microplate. Absorbance was measured at 560 nm with a microplate reader (Promega GloMax discover Multimode microplate reader). Enzyme concentration was determined by interpolation from a calibration curve prepared with the corresponding enzyme subject to the same digestion and reaction procedures. NPs encapsulation efficiency (EE%) was determined as follows: [‫]݁݉ݕݖ݊ܧ‬ே௉௦ ∙ 2.5 ‫ܧܧ‬% = ∙ 100 [‫]݁݉ݕݖ݊ܧ‬ூ௡௝ where [‫]݁݉ݕݖ݊ܧ‬ே௉௦ is the enzyme concentration in NPs and [‫]݁݉ݕݖ݊ܧ‬ூ௡௝ is the enzyme concentration in CLEAs, EAs or enzyme solution used for CLEAs NPs, EAs NPs or DE NPs synthesis, respectively. 2.5 is the dilution factor of the [‫]݁݉ݕݖ݊ܧ‬ூ௡௝ in the NPs synthesis process. 488-CLEAs 633-NPs quenching 190 µl of potassium iodide (KI) solutions at increasing concentrations (0, 100, 150, 200, 250 mM) were transferred in each well of a 96-well microplate and 10 µl of sample were added. Fluorescence was measured with a microplate reader (Promega GloMax discover Multimode microplate reader) at the following excitation and emission wavelengths: Excitation 475 nm – emission 500-550 nm for 488-CLEAs quenching Excitation 627 nm – emission 660-720 nm for 633-PLGA quenching The kinetics of the process was evaluated by applying the Stern-Volmer relationship: ‫ܨ‬଴ = 1 + ݇௤ ߬଴ [‫]ܫܭ‬ ‫ܨ‬ where ‫ܨ‬଴ and ‫ ܨ‬are the fluorescence intensities in absence and in presence of the quencher, respectively, ݇௤ is the quencher rate coefficient, ߬଴ is the lifetime of the emissive excited state of the fluorophore and [‫ ]ܫܭ‬is the quencher concentration. PPT1 CLEAs release in cuvette 200 µl of PPT1 488-CLEAs 633-NPs were transferred into a Spectra Por dialysis membrane (Biotech Cellulose Ester, molecular weight cut off 100 kDa, Spectrum Labs) and placed in 25 ml of PBS (pH 7.4) or citrate buffer (pH 4.5) at 37°C under stirring. At different time points (1, 6, 24, 48, 96, 168 and 336 hours) the external buffer was collected and replaced with 25 ml of fresh buffer. Fluorescence was measured on the external buffer aliquots with Cary Eclypse fluorometer (excitation filter 485 nm, emission filter 490-600 nm).

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Activity yield determination Enzymatic activity of CLEAs, EAs, CLEAs NPs, EAs NPs and DE NPs was determined via fluorescence-based assays by employing a specific fluorogenic substrate for each enzyme involved in this study (see Supporting Information). Activity yield (AY%) of CLEAs, EAs, CLEAs NPs, EAs NPs and DE NPs was calculated as the ratio between the specific activity of the sample (ܵ‫ܣ‬௦ ) and the specific activity of the unaltered enzyme (ܵ‫ܣ‬௖௧௥ ): ܵ‫ܣ‬௦ ‫ܻܣ‬% = ∙ 100 ܵ‫ܣ‬௖௧௥ where the specific activities are defined as unit of enzyme (U) per microgram of enzyme (ߤ݃௘ ): ܷ ܵ‫= ܣ‬ ߤ݃௘ 1 unit of enzyme (U) corresponds to 1 nmol of substrate (݊݉‫݈݋‬௦௨௕ ) hydrolized in 1 hour: ݊݉‫݈݋‬௦௨௕ ܷ= ℎ Activity expressed as U was calculated by interpolation from a calibration curve of 4-methylumbelliferone. Cell culture Mouse embryonic fibroblast cells (NIH-3T3) were purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) from Invitrogen (Carlsbad, CA). Growth medium was supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Confocal imaging NIH-3T3 cells were seeded 24 hours before the experiments into glass-bottom Petri dish (WillCo-dish GWSt-3522) to reach 80-90% confluence. Incubation with PPT1 488-CLEAs 633-NPs was performed for 1 hour at 37°C, 5% CO2 in DMEM with 10% FBS at a final NPs concentration of 1.25 mg/ml in a total volume of 200 µl. After incubation, cells were washed three times with PBS and fresh medium was added. The samples were analysed by confocal microscopy at different time points after treatment (1, 6, 24 and 48 hours). Cells were imaged using a Leica TCS SP5 SMD inverted confocal microscope (Leica Microsystems AG) interfaced with Ar and HeNe laser for excitation at 488 and 633 nm, respectively. Cells were mounted in a thermostated chamber at 37°C (Leica Microsystems) and viewed with a 40X 1.5 NA oil immersion objective (Leica Microsystems). The pinhole aperture was set to 1.0 Airy. All images were analysed using ImageJ software version 1.51 and co-localization was evaluated using JACoP plugin. The final value of Manders’ coefficient is an average obtained from the analysis of 10 images taken with the same magnification for each experiment. Enzymatic activity recovery in NCL1 cells NCL1 primary cells were seeded 24 hours before experiments into 96-well microplates to reach 80-90% confluence. Incubation with increasing doses of PPT1 CLEAs NPs, PPT1 CLEAs Tf2 NPs or PPT1 was performed for 1 hour at 37°C, 5% CO2 in DMEM with 10% FBS in a total volume of 100 µl. After incubation, cells were maintained on ice and washed three times with cold PBS. Cellular lysates were obtained by adding 30 µl of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). To evaluate enzyme activity, 15 µl of cellular lysate were added to 20 µl of substrate solution

specific for PPT1 (see Supporting Information) and incubated 2 hours at 37°C. After incubation, 150 µl of stop solution were added to each sample and fluorescence was measured with a microplate reader in a 96 well plate (Promega GloMax discover Multimode microplate reader) with excitation filter 365 nm and emission filter 415-445 nm. Total amount of protein in cellular lysate was determined via Bradford assay. Absorbance was measured at 575 nm with Promega GloMax discover Multimode microplate reader.

ASSOCIATED CONTENT Supporting Information: Supporting Tables, Figures and Experimental procedures (PDF)

CONFLICT OF INTEREST The authors declare no competing financial interest.

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Cross-Linked Enzyme Aggregates as Versatile Tool for Enzyme Delivery

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