Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging.

BACKGROUND: In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177. RESULTS: Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h. CONCLUSIONS: This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy.

Long-term biodistribution study of HPMA-ran-LMA copolymers in vivo by means of 131I-labeling.

BACKGROUND: For the evaluation of macromolecular drug delivery systems suitable pre-clinical monitoring of potential nanocarrier systems is needed. In this regard, both short-term as well as long-term in vivo tracking is crucial to understand structure-property relationships of polymer carrier systems and their resulting pharmacokinetic profile. Based on former studies revealing favorable in vivo characteristics for 18F-labeled random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and lauryl methacrylate (LMA) - including prolonged plasma half-life as well as enhanced tumor accumulation - the presented work focuses on their long-term investigation in the living organism. METHODS: In this respect, four different HPMA-based polymers (homopolymers as well as random copolymers with LMA as hydrophobic segment) were synthesized and subsequent radioactive labeling was accomplished via the longer-lived radioisotope 131I. In vivo results, concentrating on the pharmacokinetics of a high molecular weight HPMA-ran-LMA copolymer, were obtained by means of biodistribution and metabolism studies in the Walker 256 mammary carcinoma model over a time-span of up to three days. Besides, a direct comparison with the 18F-radiolabeled polymer was drawn. To consider physico-chemical differences between the differently labeled polymer (18F or 131I) on the critical micelle concentration (CMC) and the size of the polymeric micelles, those properties were determined using the 19F- or 127I-functionalized polymer. Special emphasis was laid on the time-dependent correlation between blood circulation properties and corresponding tumor accumulation, particularly regarding the enhanced permeability and retention (EPR) effect. RESULTS: Studies revealed, at first, differences in the short time (2h) body distribution, despite the very similar properties (molecular structure, CMC and size of the micellar aggregates) of the non-radioactive 19F- and 127I-functionalized polymers. Long-term investigations with the 131I-labeled polymer demonstrated that, despite a polymer clearance from the blood within 72h, there was still an increase in tumor uptake observed over time. Regarding the stability of the 131I-label, ex vivo biodistribution experiments, considering the uptake in the thyroid, indicated low metabolism rates. CONCLUSION: The observed in vivo characteristics strongly underline the EPR effect. The findings illustrate the need to combine information of different labeling approaches and in vivo evaluation techniques to generate an overall pharmacokinetic picture of potential nanocarriers in the pre-clinical setting. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENTS: The in vivo behavior of the investigated HPMA-ran-LMA copolymer demonstrates great potential in terms of an effective accumulation in the tumor.

Endocytotic uptake of HPMA-based polymers by different cancer cells: impact of extracellular acidosis and hypoxia.

BACKGROUND: Polymeric nanoparticles allow to selectively transport chemotherapeutic drugs to the tumor tissue. These nanocarriers have to be taken up into the cells to release the drug. In addition, tumors often show pathological metabolic characteristics (hypoxia and acidosis) which might affect the polymer endocytosis. MATERIALS AND METHODS: Six different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer structures (homopolymer as well as random and block copolymers with lauryl methacrylate containing hydrophobic side chains) varying in molecular weight and size were analyzed in two different tumor models. The cellular uptake of fluorescence-labeled polymers was measured under hypoxic (pO2 ≈1.5 mmHg) and acidic (pH 6.6) conditions. By using specific inhibitors, different endocytotic routes (macropinocytosis and clathrin-mediated, dynamin-dependent, cholesterol-dependent endocytosis) were analyzed separately. RESULTS: The current results revealed that the polymer uptake depends on the molecular structure, molecular weight and tumor line used. In AT1 cells, the uptake of random copolymer was five times stronger than the homopolymer, whereas in Walker-256 cells, the uptake of all polymers was much stronger, but this was independent of the molecular structure and size. Acidosis increased the uptake of random copolymer in AT1 cells but reduced the intracellular accumulation of homopolymer and block copolymer. Hypoxia reduced the uptake of all polymers in Walker-256 cells. Hydrophilic polymers (homopolymer and block copolymer) were taken up by all endocytotic routes studied, whereas the more lipophilic random copolymer seemed to be taken up preferentially by cholesterol- and dynamin-dependent endocytosis. CONCLUSION: The study indicates that numerous parameters of the polymer (structure, size) and of the tumor (perfusion, vascular permeability, pH, pO2) modulate drug delivery, which makes it difficult to select the appropriate polymer for the individual patient.

Interaction of N-(2-hydroxypropyl)methacrylamide based homo, random and block copolymers with primary immune cells.

Polymer nanoparticles (NP), e.g., polymeric micelles, represent a promising platform for drug delivery including the field of immune modulation. In respect to this potential application, identification of chemical and structural properties that affect interaction of polymers with immune cells is an important step in their preclinical characterization. A series of well-defined, fluorescently labeled homopolymers, random as well as block copolymers based on the clinically approved N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared to study the influence of polymer architecture on the interaction of polymers with primary human und murine immune cells systematically. The number average of the molar mass (M(n)) for all polymers was set to the range of 4-14 kDa with a varying ratio of hydrophilic and hydrophobic units and dispersities (D) in the range of 1.17-1.29. Cell uptake greatly depended on the polymer molecular weight and micro structure: Comparison of polymers of the same molar mass but varying ratio of hydrophilic and hydrophobic units revealed a strict dependency of cellular uptake on the size of the hydrophobic block. HPMA-ran-LMA copolymers with high amounts of lauryl side chains (15 and 20% LMA content) had highest internalization rates into human and mouse immune cells (monocytes, granulocytes, B and T cells). Our findings underline the role of particle size and composition of polymeric carriers in the field of nanomedicine.

A minimal hydrophobicity is needed to employ amphiphilic p(HPMA)-co-p(LMA) random copolymers in membrane research.

Because a polymer environment might be milder than a detergent micelle, amphiphilic polymers have attracted attention as alternatives to detergents in membrane biochemistry. The polymer poly[N-(2-hydroxypropyl)-methacrylamid] [p(HPMA)] has recently been modified with hydrophobic lauryl methacrylate (LMA) moieties, resulting in the synthesis of amphiphilic p(HPMA)-co-p(LMA) polymers. p(HPMA)-co-p(LMA) polymers with a LMA content of 5 or 15% have unstable hydrophobic cores. This, on one hand, promotes interactions of the hydrophobic LMA moieties with membranes, resulting in membrane rupture, but at the same time prevents formation of a hydrophobic, membrane mimetic environment that is sufficiently stable for the incorporation of transmembrane proteins. On the other hand, the p(HPMA)-co-p(LMA) polymer with a LMA content of 25% forms a stable hydrophobic core structure, which prevents hydrophobic interactions with membrane lipids but allows stable incorporation of membrane proteins. On the basis of our data, it becomes obvious that amphiphilic polymers have to have threshold hydrophobicities should an application in membrane protein research be anticipated.

HPMA-LMA copolymer drug carriers in oncology: an in vivo PET study to assess the tumor line-specific polymer uptake and body distribution.

Polymeric drug carriers aim to selectively target tumors in combination with protecting normal tissue. In this regard polymer structure and molecular weight are key factors considering organ distribution and tumor accumulation of the polymeric drug delivery system. Four different HPMA based copolymer structures (random as well as block copolymers with lauryl methacrylate as hydrophobic block) varying in molecular weight, size and resulting architecture were analyzed in two different tumor models (AT1 prostate carcinoma and Walker-256 mammary carcinoma) in vivo. Polymers were labeled with (18)F and organ/tumor uptake was followed by µPET imaging and ex vivo biodistribution. Vascular permeability was measured by dextran extravasation and vascular density by immunohistochemistry. Cellular polymer uptake was determined in vitro using fluorescence-labeled polymers. Most strikingly, the high molecular weight HPMA-LMA random copolymer demonstrated highest tumor uptake and blood pool concentration. The molecular structure (e.g., amphiphilicity) is holding a higher impact on desired in vivo properties than polymer size. The results also revealed pronounced differences between the tumor models although vascular permeability was almost comparable. Accumulation in Walker-256 carcinomas was much higher, presumably due to a better cellular uptake in this cell line and a denser vascular network in the tumors. These investigations clearly indicate that the properties of the individual tumor determine the suitability of polymeric drug carriers. The findings also illustrate the general necessity of a preclinical screening to analyze polymer uptake for each individual patient (e.g., by noninvasive PET imaging) in order to individualize polymer-based chemotherapy.

PEGylation of HPMA-based block copolymers enhances tumor accumulation in vivo: a quantitative study using radiolabeling and positron emission tomography.

This paper reports the body distribution of block copolymers (made by controlled radical polymerization) with N-(2-hydroxypropyl)methacrylamide (HPMA) as hydrophilic block and lauryl methacrylate (LMA) as hydrophobic block. They form micellar aggregates in aqueous solution. For this study the hydrophilic/hydrophobic balance was varied by incorporation of differing amounts of poly(ethylene glycol) (PEG) side chains into the hydrophilic block, while keeping the degree of polymerization of both blocks constant. PEGylation reduced the size of the micellar aggregates (Rh=113 to 38 nm) and led to a minimum size of 7% PEG side chains. Polymers were labeled with the positron emitter (18)F, which enables to monitor their biodistribution pattern for up to 4h with high spatial resolution. These block copolymers were investigated in Sprague-Dawley rats bearing the Walker 256 mammary carcinoma in vivo. Organ/tumor uptake was quantified by ex vivo biodistribution as well as small animal positron emission tomography (PET). All polymers showed renal clearance with time. Their uptake in liver and spleen decreased with size of the aggregates. This made PEGylated polymers--which form smaller aggregates--attractive as they show a higher blood pool concentration. Within the studied polymers, the block copolymer of 7% PEGylation exhibited the most favorable organ distribution pattern, showing highest blood-circulation level as well as lowest hepatic and splenic uptake. Most remarkably, the in vivo results revealed a continuous increase in tumor accumulation with PEGylation (independent of the blood pool concentration)--starting from lowest tumor uptake for the pure block copolymer to highest enrichment with 11% PEG side chains. These findings emphasize the need for reliable (non-invasive) in vivo techniques revealing overall polymer distribution and helping to identify drug carrier systems for efficient therapy.

Modifying the body distribution of HPMA-based copolymers by molecular weight and aggregate formation.

There is a recognized need to create well-defined polymer probes for in vivo and clinical positron emission tomography (PET) imaging to guide the development of new generation polymer therapeutics. Using the RAFT polymerization technique in combination with the reactive ester approach, here we have synthesized well-defined and narrowly distributed N-(2-hydroxypropyl)methacrylamide homopolymers (pHPMA) (P1* and P2*) and random HPMA copolymers consisting of hydrophilic HPMA and hydrophobic lauryl methacrylate comonomers (P3* and P4*). The polymers had molecular weights below (P1* and P3*) and above the renal threshold (P2* and P4*). Whereas the homopolymers dissolve in isotonic solution as individual coils, the random copolymers form larger aggregates above their critical micelle concentration (∼ 40 nm), as determined by fluorescence correlation spectroscopy. Structure-property relationships of the pharmacokinetics and biodistribution of the different polymer architectures were monitored in the living organism following radiolabeling with the positron emitter (18)F via fluoroethylation within a few hours. Ex vivo organ biodistribution and in vivo µPET imaging studies in male Copenhagen rats revealed that both size and the nature of the aggregate formation (hydrophobically modified copolymers) played a major role in blood clearance and biodistribution, especially concerning liver and kidney accumulation. The high-molecular-weight random copolymer P4* (hydrophobically modified), in particular, combines low liver uptake with enhanced blood circulation properties, showing the potential of hydrophobic interactions, as seen for the represented model system, that are valuable for future drug carrier design.

HPMA based amphiphilic copolymers mediate central nervous effects of domperidone.

In this study we give evidence that domperidone encapsulated into amphiphilic p(HPMA)-co-p(laurylmethacrylate) (LMA) copolymer aggregates is able to cross the blood-brain barrier, since it affected motor behaviour in animals, which is a sensitive measure for CNS actions. Carefully designed copolymers based on the clinically approved p(HPMA) were selected and synthesized by a combination of controlled radical polymerization and post-polymerization modification. The hydrodynamic radii (R(h) ) of amphiphilic p(HPMA)-co-p(LMA) alone and loaded with domperidone were determined by fluorescence correlation spectroscopy.

Radioactive labeling of defined HPMA-based polymeric structures using [18F]FETos for in vivo imaging by positron emission tomography.

During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.