Multistage Passive and Active Delivery of Radiolabeled Nanogels for

Article pubs.acs.org/Biomac

Multistage Passive and Active Delivery of Radiolabeled Nanogels for Superior Tumor Penetration Efficiency Natascha Drude,†,‡,§ Smriti Singh,‡,§ Oliver H. Winz,† Martin Möller,*,‡ Felix M. Mottaghy,*,†,∥ and Agnieszka Morgenroth† †

Department of Nuclear Medicine, RWTH Aachen University, 52074 Aachen, Germany DWI − Leibniz-Institute for Interactive Materials, RWTH Aachen University, 52074 Aachen, Germany ∥ Department of Nuclear Medicine, Maastricht University Medical Centre, 6229 HX Maastricht, Netherlands ‡

S Supporting Information *

ABSTRACT: Development of nanosized drug delivery systems in cancer therapy is directed toward improving tumor selectivity and minimizing damages of healthy tissue. We introduce a delivery system with synergistic optimization and combination of passive and active targeting strategies. The approach is based on radiopeptide labeled redox sensitive hydrophilic nanogels, which exploit passive targeting by the enhanced permeability and retention effect while avoiding elimination by the mononuclear phagocyte system and fast hepatic and renal clearance. The targeting peptide promotes endocytotic uptake of the nanogels by cancer cells. Specific to this delivery system, tumor-specific degradation by the antioxidant glutathione enhances penetration and retention within the tumor tissue. Using in vivo molecular imaging we demonstrate the superiority of combined passive and active targeting with down-sizable nanogels over exclusive passive targeting. Furthermore, the homogeneous tumor distribution of functionalized nanogels compared to the clinically used mere radiopeptide supports the potentially high impact of our targeting concept.

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INTRODUCTION In cancer therapy, multifunctional nanoparticles combining therapeutic agents, molecular targeting, and diagnostic imaging capabilities are emerging as the next generation of multifunctional nanomedicine.1−3 Primarily, this concept enables targeting and control of the delivery of a marker or therapeutic agent to the tumor tissue. The active targeting relies on a ligand that binds specifically to receptors overexpressed by cancer cells. Passive targeting exploits variations in the blood vessels permeability, which is specific to the size and properties of the nanoparticles. For a successful application, both active and passive targeting must go hand in hand as the active targeting interaction between the cell and the nanoparticle requires a presynaptic passive delivery to the tumor tissue. In order to hit the tumor cells the particulate drug delivery system should additionally overcome the barriers of systemic circulation, i.e. early elimination by the mononuclear phagocyte system (MPS) as well as fast hepatic and renal elimination. At the same time, it should efficiently accumulate in the tumor tissue and penetrate it deeply. Proper size and hydrophilic modification provide the means to evade renal filtration and to prevent their elimination by MPS,4,5 thus, enabling long circulation time in the blood in order to enhance the particle’s opportunity to interact with the tumor tissue. Besides the improved systemic availability, the long circulation time © 2017 American Chemical Society

supports the accumulation of nanoparticles in the tumor by the enhanced permeability and retention effect (EPR).6 The EPR effect is based on leakiness of the tumor vasculature combined with poor lymphatic drainage. Thus, nanoparticles of a size between 10−500 nm can selectively extravagate into tumor tissue. In contrast, low molecular weight drugs diffuse nonselectively through the endothelial layer of normal tissues, inducing significant off-target toxicity at therapeutic doses. However, the EPR effect has its own limitations, that is, the tremendous heterogeneity in tumor vessel leakiness over space, time, and different types of tumors. Furthermore, increased interstitial fluid pressure limits the transport through the dense collagen matrix surrounding the tumor. Thus, the insufficient tumor penetration still remains a key challenge for particle-mediated drug delivery systems.7,8 To enhance tumor penetration stimuli sensitive nanoformulations designed to shrink and degrade in response to the different conditions, for example, lower pH and different redox potential of tumor microenvironment are extensively studied.8−14 Received: May 2, 2017 Revised: June 23, 2017 Published: July 6, 2017 2489

DOI: 10.1021/acs.biomac.7b00629 Biomacromolecules 2017, 18, 2489−2498

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Figure 1. Illustration of the multistep concept of tumor targeting with peptide functionalized redox sensitive hydrophilic nanogels. In a first step, the large highly water swollen polymeric network passively diffuses through the leaky vasculature and squeezes through the collagen matrix; in a second step, close to the tumor microenvironment nanogels start to degrade into low molecular weight prepolymers with covalent attached targeting peptide. Upon intra- and extracellular degradation of the functionalized nanogel, the radiolabeled peptide is released and can actively target receptor expressing tumor cells. The intact nanogel prevents accumulation in healthy tissue, while the degraded structures enhance tumor penetration and the targeting peptide improves tumor retention.

uptake by the tumor cells can improve the retention and the intratumoral distribution of the radionuclide. As a consequence, the EPR effect, receptor mediated targeting, and radionuclide therapy can be combined in a synergistic way toward significant improvement of tumor targeting and enhancement of tumor cytotoxicity. Based on these strategies, we demonstrate hydrophilic nanogels as a radionuclide carrier with highly improved tumor penetration ability as a result of synergistic combination of passive and active targeting strategies. Until now, the option of radiolabeling of nanogels for targeting tumors has only scarcely been evaluated.22,23 By this work, we developed a multistage concept with biodegradable nanogels for improved delivery of endogenous ionizing radiation. In the first step, size driven accumulation due to the EPR effect takes place. In a second process step, degradation of the nanogels within the tumor (i.e., in the interstitium as well as in the tumor cells) releases the small radioisotope−peptide complexes and boosts their deep penetration into the tumor. In a third and final step, specific uptake by the tumor cells allows longer retention of radioisotope−peptide complexes in the tumor tissue (Figure 1).

Considering these facts, the maximization of the penetration potential of the active agent within the tumor interstitium is mandatory to attain the desired therapeutic potential.15−17 Endogenous radiotherapy offers high therapeutic efficiency due to the direct radiation delivery to the tumor, which results in an induction of tremendous cytotoxic effects. Advantageously, due to the high therapeutic efficiency in situ for endogenous radiotherapy, only a very low loading capacity of the carrier is needed, which is a general limiting factor for molecular drugs. Yet, endogenous radiotherapy is still limited in its use due to restricted targeting potential and undesired side effects, thus, only few endogenous radiotherapy approaches are implemented in standard patient treatment protocols.18,19 Nanogels offer a smart multistage delivery formulation which can overcome these current limitations of solid nanoparticle approaches. The open soft structure of hydrophilic nanogels enables long circulation time and flexible presentation of ligated cell receptor targeting peptides.16,20 Next to a superior circulation time, the compliance of the gel structure is favorable for perfusion into the tumor as the gels can squeeze themselves into smaller holes. Importantly, active agents and the tumor targeting ligands can be linked covalently in order to prevent early release. However, the stealth character and compliance of the soft hydrophilic nanogels is preserved only if the modification is minimal. Importantly, the concept of using nanogels for fractionated application of endogenous irradiation emitters may benefit from improved passive delivery. The preconditioning by means of external radiotherapy was shown to enhance penetration and to improve the intratumoral distribution of nanostructures by enhancing the EPR effect.21 The cytotoxic action of a radionuclide depends on its local concentration, retention, and its decay time in situ. Moreover, the combination with a targeting peptide that actively affects

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EXPERIMENTAL METHODS

The basic structural element of the nanogels is a six arm star shaped poly(ethylene glycol) based polymer with 90% ethylene oxide (EO) and 10% propylene oxide (PO), which is thiol functionalized (sPEGSH) for attaching either maleimide-NODAGA, maleimide-DOTATATE (malDOTA-TATE (co. Caslo)), and Alexa Fluor 488maleimide. Detailed information on synthesis and characterization methods are described in earlier publications24 and the Supporting Information (Experimental procedures). Functionalized prepolymers are then used for the synthesis of nanogels suitable for nuclear molecular imaging via inverse miniemulsion technique (for details, see Supporting Information, Experimental procedures).25 Nanogels could 2490

DOI: 10.1021/acs.biomac.7b00629 Biomacromolecules 2017, 18, 2489−2498

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Biomacromolecules Scheme 1a

a

(A) Nanogels are synthesized from six-arm, star-shaped poly(ethylene glycol) polymer with 90% ethylene oxide (EO) and 10% propylene oxide (PO) with thiol end groups (sPEG-SH). (B) Functionalization of thiolated prepolymer (sPEG-SH) with maleimide-NOTA chelator. (C) Functionalization of thiolated prepolymer (sPEG-SH) with maleimide-DOTA-TATE (malDOTA-TATE). The chelator functionalized thiolated prepolymers were used for nanogels synthesis followed by radiolabelling with 68Ga (t1/2 = 68 min). (D) Structure of the free radiopeptide for bench marking. acquisition started with (for first kinetic studies) or 5−10 min after intravenous injection of the radiolabeled compound. Acquisition time was 1 h and after data acquisition, Positron emission tomography (PET) images were reconstructed by a three-dimensional orderedsubsets expectation maximum (OSEM) algorithm. All data were corrected for attenuation, scatter, dead time, and decay. Ex vivo γcounting results are expressed as mean ± SD. Statistical calculations for in vitro and in vivo experiments were performed using OriginPro 8.5.1 for Windows. Effects were considered to be statistically significant if p ≤ 0.05.

be redispersed in H2O without the addition of surfactant due to the stabilizing effect of the polymer chain. When redispersing the nanogels in water a swelling is observed, the extent of which is inversely proportional to the degree of cross-linking of the nanogels. Cryo-field emission scanning electron microscopy (cryo-FESEM), dynamic light scattering (DLS), and ultrahigh resolution FESEM were used to determine the morphology of the newly developed nanogels in swollen and in dry state, respectively (for details, see Supporting Information, Experimental procedures). Radiolabeling using no carrier added (n.c.a.) [68Ga]Cl3 was performed without destruction of the polymers and nanogels. In total, 5 male Wistar rats and 10 balb/c nude mice were used for the presented in vivo experiments. Rats as well as mice were housed under standardized conditions with a light/dark cycle of 12 h/12 h at 22 ± 2 °C with a relative air humidity range of 30−70%. Animals received Ssniff diet (K−H, Ssniff, 59494 Soest, Germany). For water, supply bottles were used and filled with sterile water that was pH reduced. Mice were randomized to receive either [68Ga]DOTATATE-nanogels, [68Ga]NOTA-nanogels, and as clinical gold standard [68Ga]DOTA-TATE as a control. All injections were performed intravenously into the tail vein under isoflurane narcosis. The data

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RESULTS AND DISCUSSION Synthesis and Chemical Characterization of Nanogels. The nanogels have been synthesized from six-arm, starshaped poly(ethylene glycol) polymer with 90% ethylene oxide (EO) and 10% propylene oxide (PO) with thiol end groups (sPEG-SH), by cross-linking the prepolymers in inverse miniemulsion via oxidation (Scheme 1).25 Importantly, the thiol functionalization facilitates further modification of the prepolymer by either maleimide-NOTA (2,2′-(7-(2-((2-(2,52491

DOI: 10.1021/acs.biomac.7b00629 Biomacromolecules 2017, 18, 2489−2498

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Biomacromolecules

Figure 2. Synthesis and structural characterization of nanogels. (A) Angle-dependent measurement of the nanogels in the swollen state by DLS with determination of the diffusion coefficient via linear regression. (B) Representative size distribution function (angle 90°); PDI estimated by dynamic light scattering (DLS) < 0.25. (C) Example of the Guinier Plot of a NOTA-nanogel results in a radius of gyration of Rg = 194 nm. (D) Nanogel image on the silicon wafer taken by ultrahigh resolution FESEM (SU9000; in dry state) and (E) nanogels in water (swollen state) by cryo-FESEM. (F) EDX-mapping of the chemical elements carbon (green), oxygen (blue), and Gallium (red) is shown, respectively. (G) EDX-spectrum demonstrates a 2−3 wt % content of Ga. The Si-peak in the spectrum originates from the silicon wafer used to mount the nanogels. Very small signals of Ca and P originate from the buffer used during the labeling experiment.

scattering experiments (Figure S1C−E). This indicates a homogeneous peptide distribution within the nanogel. FESEM-EDX imaging affirms the open structure of the nanogel-network and the unhampered accessibility of the chelator in the polymeric construct for radiolabeling. Gallium is evenly distributed throughout the entire gel structure and exclusively traceable in the nanogel, which confirms the efficiency of Ga3+ binding within the nanogels (Figure 2D− G). This again indicates that the peptide is not on the surface of the nanogel but rather homogeneous distributed throughout the gel structure. Internalization and Stability of Nanogels. The cytotoxicity of the nanogels was evaluated in THP-1 monocytes, PMA stimulated THP-1 macrophages and in the AR42J cancer cells (Figure S2). Regardless of the concentrations and the incubation periods tested, the viability of all cell lines remained unaffected. Using these cell lines, we investigated the stealth characteristic of the star PEG-based nanogels in vitro. Monocytes did not internalize the hydrophilic nanogels. Contrary, the highly active endocytotic macrophages showed an efficient size and concentration dependent nanogel engulfment.24 Similarly, due to the elevated endocytotic activity as a general marker of almost all cancer cells,29 the epithelial pancreas cancer cells AR42J exhibited an effective time and concentration-dependent nanogel uptake (Figure S3). Reductive degradation of the nanogels was demonstrated by means of Alexa Fluor 488-labeled derivatives as described previously.24 Incubation of fluorescence-labeled nanogels (0.5 mg/mL) for at least 1 h with 10 mM of glutathione (GSH) led to extensive particle degradation as detected by fluorescence microscopy, a native Page analysis and DLS (Figure 3B−E). The original 200−500 nm nanogels underwent a size reduction to about 3.5 nm, which corresponds to the size of the single prepolymers (Figure 3A).

dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)1,4,7-triazonane-1,4-diyl)diacetic acid), maleimide-DOTATATE (2,2′,2″-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)ethyl)-2-oxo)amino) pentyl)-amino)-2-oxoethyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetic acid-(Tyr3)-octreotate) (malDOTA-TATE), and Alexa Fluor 488-maleimide. The successful conjugation of PEG polymers was confirmed by 1 H NMR analysis (Figure S1). The disulfide bridge linkages between the prepolymers can be broken in a reducing environment. This leads to degradation of the nanogels and finally to release of the lower molecular weight building blocks. Full characterization of the nanogel structure was achieved by cryo-scanning electron microscopy (Cryo-FESEM), size exclusion chromatography (SEC), light scattering, and zeta potential. The average size of nanogels was between 200 and 500 nm with a polydispersity index (PDI) of ≤1.3. This diameter is an appropriate size for the highly flexible constructs to preferentially accumulate in the tumor through the EPR effect since the gaps between adjacent endothelial cells in the tumor vessels ranges between 380 and 780 nm.26,27 On the other hand, the nanogels are large enough to avoid rapid clearance from the circulation through renal filtration which is characteristic for solid nanoparticles smaller than 5 nm.28 Combined DLS/SLS analysis of the nanogels gave the hydrodynamic radius Rh and the radius of gyration Rg (Figure 2A−C). The ratio of the two radii confirmed a spherical shape of the gels (Rg/Rh = 0.78). Importantly, the structure remains stable under labeling conditions at room temperature as well as at 95 °C (Figure S1B). The attachment of the peptide had no influence on either size of the nanogels (malDOTA-TATENG) or their morphology or zeta potential (−10 mV < ζ > −14 mV) compared to the nonfunctionalized gels (NOTA-nanogels) as additionally demonstrated in microscope and light 2492

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sample (retrobulbar) as well as urine (puncture of the bladder) were collected 7 h post-injection. In the interval between injection and sample collection, animals were awake, thus, some radioactivity was already voided and the urine sample will not be an exact quantitative measure of the amount of renal excretion, but gives a view on the excreted metabolites/nanogel fragments. The blood sample was centrifuged and serum was qualitatively analyzed for intact nanogels and for prepolymers via native page with phosphor imaging. The sample isolated from the blood showed only minor degradation of 20−25% relative to the amount of intact nanogel in the plasma, while in the urine and in the sample incubated in vitro with GSH solely the prepolymer fraction was detectable (Figure 3E). Pharmacokinetic Study in Healthy Animals. For in vivo studies, the nanogels were first conjugated with a macrocyclic chelator and then radiolabeled with the positron emitting radionuclide 68Ga. Competition experiments demonstrated efficient radiolabeling of chelator conjugated nanogels with 68 Ga (Figure S5). Biodistribution and clearance kinetics of 68 Ga-labeled nanogels ([68Ga]NOTA-nanogels) were evaluated in healthy Wistar rats after intravenous injection. The small animal positron emission tomography (μPET) dynamically acquired over the 2 h after injection indicated a very high level of remaining radioactivity in the blood pool at the end of the acquisition (Figures 4A and S5). However, it must be noted that within the first 15 min the nanogel activity in the blood decreased by 15% (Figure 4B). After this first elimination, the nanogel concentration in the blood pool decreased only marginally, which is not observed with standard radiolabeled peptides. The initial fast loss of about 15% in activity may be explained by the size distribution, where the smaller nanogels in connection with their high shape flexibility may escape the system via renal elimination. The activity in the kidneys and the bladder accounted for ≤15% of the total injected nanogels (Figure 4B). The phase of slower elimination is consistent with a continuous redistribution of nanogels between the vascular and extravascular compartments in connection with a slow degradation in the bloodstream caused by disulfide exchange.33−35 No significant accumulation in liver or spleen was observed confirming low uptake of nanogels by MPS and the expected long circulation of the nanogels was demonstrated (Figure 4C). In contrast, previous in vivo studies with more compact nanoparticles have demonstrated their fast elimination from the blood pool via MPS and hepatic extraction.36 In Vitro Characterization of Nanogels as Radiolabeled Tumor Targeting Delivery Systems. Improved intratumoral retention and increased uptake by the tumor cells was addressed by endowing the nanogels with a tumor targeting potential. To this end, the redox-sensitive nanogels were functionalized with a mainly somatostatin receptor subtype 2 (SstR2) addressing peptide.37 Recent studies have demonstrated selective and efficient chemotherapeutic drug delivery potential of octreotide or lanreotide peptide functionalized micelles to SstR2 expressing tumor cells.38,39 Compared to the clinically used radiopeptide precursor DOTA-TATE the herein used malDOTA-TATE (octreotide) has an additional lysine function with maleimide functionality (marked red in Scheme 1). This modification allowed for covalent binding of the peptide to the prepolymer respectively to the nanogel as required for subsequent radiolabeling with 68Ga and as required for tumor-specific targeting. However, compared to the published values after 4 h of incubation, the uptake of the

Figure 3. Reductive degradation of nanogels by tripeptide glutathione. (A) Angle-dependent DLS analysis of nanogels before (squares) and after (circles) degradation. (B, C) Fluorescence microscope images of reductive degradation of redox sensitive nanogels by 10 mM GSH (100-fold magnification) before reduction (B) and after reduction (C). (D) Native SDS Page analysis of untreated and GSH-treated Alexa Fluor-labeled nanogels (450 μg of nanogel per Lane). (E) Native Page and phosphor imager analysis of 68Ga-nanogel (20 kBq per lane). Intact nanogels were detected at high molecular size (>250 kDa) reduced nanogels appeared at 10−50 kDa. Samples were taken from plasma at 7 h post-injection and from urine at 7 h post-injection.

The degradation of the nanogels in the tumor tissue is supposed to facilitate a deeper penetration into the tumor parenchyma of the in situ regenerated lower molecular weight prepolymers.30,31 The preferential intratumoral nanogel degradation is induced by the increased redox potential in malignant tissues. Extracellular fluids within the tumor interstitium show an increased presence of GSH (10 to 100-fold compared to healthy tissue). The detected GSH concentration in the isolated AR42J tumor cells was at least four times higher than in normal cells or in blood serum32 (Figure S4). The in vivo stability of nanogels was evaluated in the blood and in the urine samples. 68Ga-labeled nanogels (200 μL, c = 1.0−1.5 μg/μL with an activity of 10−15 MBq) were i.v. injected into AR42J xenografted balb/c mice and a blood 2493

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Figure 4. General biodistribution of radiolabeled nanogels in healthy animals. (A) μ-PET image of a nonfunctionalized [68Ga]NOTA-nanogel i.v. injected healthy Wistar rat 2 h post-injection (reconstruction 3D-OSEM; Maximum Intensity Projection (MIP)); (B) kinetic profile: first hour time activity analysis of [68Ga]NOTA-nanogel in kidneys (open square), heart (circle), and the whole body (exclusive heart and kidneys; triangle); (C) γcounter analysis of accumulation of [68Ga]NOTA-nanogel in % of injected dose per gram tissue (4 h p.i.). Error bars represent standard deviation (n = 3). After 4 h p.i., highest activity is still detected in the blood pool.

where the targeting peptide is homogeneously distributed within the gel. High hydrophilicity of ultrasoft nanogels and homogeneous distribution of active peptides can initially slow down the nanogels−cancer cell interaction but the elevated extracellular GSH level initiates the nanogel degradation, which retrieves the peptides and thereby supports the cellular uptake. In Vivo Characterization of Nanogels as Radiolabeled Tumor Targeting Delivery Systems. First investigations of the radioactive labeled nanogels ([68Ga]NOTA-nanogels) in AR42J xenografted balb/c nude mice showed a different pharmacokinetic profile compared to healthy animals. We evaluated the optimal amount of peptide functionalization according to general biodistribution studies in healthy animals and found that higher peptide amount (4 wt %) led to an increased uptake in the liver and spleen. Thus, for xenografted animal studies we compared and studied those two nanogels with closest biodistribution (without peptide (NOTA-nanogel) and with 1 wt % peptide (malDOTA-TATE-nanogel), which did not show significant difference in tissue accumulation in healthy animals (Figure S7). After the first initial elimination of nanogels through the renal system observed in healthy animals, in xenografted mice an additional slow renal excretion occurred 3−4 h post-injection. This activity decrease can be seen as a prolonged passive uptake into the tumor which might be explained by early degradation of the radiolabeled nanogels due to the different redox potential caused in tumor environment and thereby a release of degraded gel structures. Tumor selective nanogel degradation arises from the altered redox homeostasis of tumor cells leading to an increased concentration of thiols like glutathione within the tumor cells as well as in the surrounding tissue.34 Blood samples and HPLC analysis proved the degradation of the particles in plasma and urine samples (Figure S8). Interestingly, this effect was not seen for peptide functionalized [ 68 Ga]malDOTA-TATE-nanogels. Upon degradation, the [68Ga]malDOTA-TATE-nanogels seem to be retained due to the active targeting of the [68Ga]malDOTA-TATE-prepolymer (Figure 6A). The time activity curves for both tumors show an initial rather comparable accumulation in the tumor, while after 4 h, the radioactivity in the tissue decreases to a minimum for the unfunctionalized ([68Ga]NOTA-nanogels, whereas a slight increase is observed for the functionalized [68Ga]malDOTATATE-nanogels (Figure 6B). The acquisition started about 10

mere modified peptide malDOTA-TATE is by 23% lower than that of the clinically used DOTA-TATE.40 The ability of functionalized radiolabeled nanogels (malDOTA-TATE-nanogel) to address the SstR2 expressing cells was studied in vitro in AR42J cells. The radiolabeled nanogels without the targeting peptide (NOTA-nanogel) and the free radiopeptide (not ligated to the nanogels; malDOTA-TATE) served as a control. As expected, the free radiopeptide [68Ga]malDOTA-TATE showed the highest uptake due to the accessibility of the targeting peptides to the SstR2 receptors (Figure 5). The direct

Figure 5. Cellular uptake of unfunctionalized nanogel (NOTAnanogel) and peptide-functionalized nanogel (malDOTA-TATEnanogel). SstR2-espressing AR42J cells were incubated for 1 and 4 h with 68Ga-labeled unfunctionalized (NOTA-nanogel) and functionalized (malDOTA-TATE-nanogel) as well as with nonencapsulated radiolabeled malDOTA-TATE derivative and clinically used DOTATATE. Error bars represent standard errors (n = 3). The malDOTATATE derivative shows a lower uptake by AR42J cells than the clinically used DOTA-TATE (32.7% ± 1.6%). This equals 23% lower uptake of our derivative compared to the clinically used peptide. The DOTA-TATE uptake after 4 h of incubation corresponds to the literature value40 (*p < 0.05; **p < 0.01; ***p < 0.001).

comparison of NOTA-nanogels with malDOTA-TATE-nanogel clearly indicated a significantly enhanced uptake of targeting ligand conjugated nanogels by the tumor cells. The longer the incubation time of nanogels with the malignant cells, the higher was the difference in cell uptake of unfunctionalized NOTAnanogels compared to malDOTA-TATE-nanogels. For NOTAnanogels no significant increase of uptake over the incubation time was detected, while the uptake of malDOTA-TATE nanogels raised by more than 100% . This time-dependent uptake profile confirms the structural concept of nanogel, 2494

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Biomacromolecules Figure 6. continued

xenografted nude mice injected with [68Ga]malDOTA-TATE-nanogel (black bars) and [68Ga]NOTA-nanogel (red bars) in % of injected dose per gram tissue (6.5 h p.i.) shows 50% higher tumor uptake in case of [68Ga]malDOTA-TATE-nanogel compared to only EPR driven accumulation by the [68Ga]NOTA-nanogel. Error bars represent standard deviation (n = 4); *p < 0.05.

min after i.v. injection. The activity values were corrected for the injected activity and the activity decay of the 68Ga(III) ions. The tumor accumulation at later points in time was investigated and compared in an in vivo biodistribution study combined with postmortem analysis in AR42J xenografted balb/c nude mice. A significantly improved tumor accumulation of the malDOTA-TATE-nanogels was observed 6.5 h p.i. compared to unconjugated NOTA-nanogels (8.2% ± 1.4% ID/ g vs 4.1% ± 1.2% ID/g). Importantly, there was no statistical significant increase in hepatic clearance for malDOTA-TATEnanogels compared to NOTA-nanogels without peptide (Figure 6C). For both nanogels uptake by SstR2 expressing healthy organs like, for example, spleen or pancreatic β-cells26 was negligible. It is noteworthy that the malDOTA-TATEnanogels displayed a highly increased accumulation and retention in two very small xenografts (