Surfactant free preparation of biodegradable dendritic polyglycerol nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceutical biomacromolecules

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Abstract

In this paper we report a novel approach to generate biodegradable polyglycerol nanogels on different length scales. We developed a mild, surfactant free inverse nanoprecipitation process to template hydrophilic polyglycerol nanoparticles. In situ crosslinking of the precipitated nanoparticles by bioorthogonal copper catalyzed click chemistry allows us to obtain size defined polyglycerol nanogels (100–1000 nm). Biodegradability was achieved by the introduction of benzacetal bonds into the net points of the nanogel. Interestingly, the polyglycerol nanogels quickly degraded into low molecular weight fragments at acidic pH values, which are present in inflamed and tumor tissues as well as intracellular organelles, and they remained stable at physiological pH values for a long time. This mild approach to biodegradable polyglycerol nanogels allows us to encapsulate labile biomacromolecules such as proteins, including the therapeutic relevant enzyme asparaginase, into the protein resistant polyglycerol network. Enzymes were encapsulated with an efficacy of 100% and after drug release, full enzyme activity and structural integrity were retained. This new inverse nanoprecipitation procedure allows the efficient encapsulation and release of various biomacromolecules including proteins and could find many applications in polymer therapeutics and nanomedicine.

Introduction

Therapeutically relevant proteins such as antibodies, cytokines, growth factors, and enzymes play an increasing role in the treatment of viral, malignant, and autoimmune diseases [1], [2]. Therapeutic proteins, however, often suffer from insufficient stability and shelf-life, costly production, immunogenic and allergic potential, as well as poor bioavailability and sensitivity towards proteases [3]. An elegant method to overcome most of these problems is the attachment of polyethylene glycol (PEG) chains onto the surface of the protein [4], [5], [6], [7]. Covalent PEGylation of the native protein increases its molecular weight and as a result prolongs the half-life in vivo [3]. By molecular weight elevation passive targeting to solid tumors can be achieved due to the enhanced permeation and retention effect [8], [9]. Additionally, the resistance to proteases is increased by the PEG coating. PEGylation of proteins, however, still suffer from the loss of biological activity [5].

These problems can be circumvented, when proteins are encapsulated non-covalently into nanogels [10], [11]. Nanogels, which are hydrogel particles on the nanometer scale, are highly water swollen scaffolds that exhibit similar properties as many biological objects, thus making them excellent candidates for biomedical applications [12], [13], [14], [15], [16]. Within the past few years, two strategies have evolved for the encapsulation of biomacromolecules into nanogels. They can be encapsulated after nanogel formation by the diffusion of the guest into the nanogel due to specific interactions with the nanogels [17]. Strong interactions with the gel matrix, however, might cause denaturation of the payloads and diffusion limitations lead to low encapsulation efficiencies. Another strategy entraps the payloads in-situ during the nanogel formation process, which ensures high encapsulation efficiencies and a homogenous distribution of the guest within the entire gel particle [11]. Additionally, the encapsulated payloads can be embedded very tightly in the gel matrix by tuning the degree of crosslinking. Thus, the guest can be transported to the target site without any loss of payload by leaching.

For drug release, nanogels require external stimuli to achieve a controlled release of the guest at the target site. Guest liberation by nanogel degradation and resulting network dissolution is the most promising strategy. Release kinetics can be tuned by the degradation kinetics and the generated low molecular weight degradation fragments can be cleared by the kidneys, which reduces the possibility of long-term toxicity due to organ accumulation [5]. Various chemical bonds such as disulfides [18], [19], acetals [20], [21], ketals [22], [23], phosphate esters [24], silyl ethers [25], and esters [26] have been introduced into nanogel networks which are cleaved in response to specific biological stimuli including pH or reductive environments [27].

In-situ encapsulation, however, requires mild nanogel preparation conditions to retain biomacromolecule activity, especially when dealing with sensitive substances such as proteins. Nanogels are usually prepared by the templation of reactive monomers on the nanometer scale and subsequent crosslinking of the templates to obtain hydrogel nanoparticles. The most frequently used methods are templations in mini [28], [29], [30], [31], [32], [26]- and microemulsion droplets [33], [34], [35]. High energy input by ultrasonication, which is required for the formation of miniemulsions, does not allow the encapsulation of labile compounds by this technique. The formation of microemulsions requires high surfactant loadings, which lead to purification problems and thereby also limit the applicability of this technique.

Another approach which has been developed by Whitesides et al. [36] and was further improved by the De Simone group [37] is the templation of polymer macromonomers in soft lithography templates. Clean room conditions, however, are required for this approach possibly limiting the broad application of this technique. Another widely used approach is based on the crosslinking of polymeric micelles [15]. Even though this approach is often called “surfactant free” in literature, amphiphilic polymers are required which might interact and denature the encapsulated payloads. Additionally, material parameters like size, elasticity and shape are difficult to influence.

The nanoprecipitation technique has evolved as a powerful tool for the preparation of hard polymer nanoparticles built from polystyrene (PS) [38], polymethyl methacrylate) (PMMA) [39], [40], and polylactic acid/polylactic-co-glycolic acid (PLA/PLGA) [41], [42], [43]. Particles prepared by nanoprecipitation avoid the above-mentioned downsides and allows hydrophobic drugs to be encapsulated [43]. To our knowledge, however, the nanoprecipitation technique has not been applied for the preparation of nanogels, made from hydrophilic polymers.

In this paper we describe for the first time the templation of hydrophilic polyglycerol-macromonomers by nanoprecipitation and subsequent chemical crosslinking of the precipitated particles. We use dendritic polyglycerol (dPG) as nanogel scaffold material, due to its outstanding multifunctionality [44], [45] and protein resistance [46], [47], [48]. Minimal interaction with the polymer matrix provides maximal protein stabilization. Additionally, the rigidity of dendritic macromolecules generates high diffusion barriers for the encapsulated proteins, thereby facilitating stable transport behavior. We call this novel approach inverse nanoprecipitation due to the inversion of polarity in the nanoprecipitation process. The combination of the mild nanogel preparation method with the biocompatible nature of polglycerol material allows us to encapsulate therapeutic enzymes with encapsulation efficacies higher than 99% under full retention of activity and structural integrity. Finally we show that the incorporation of pH labile, cyclic benzacetal bonds into the nanogel network leads to degradation triggered controlled enzyme release at acidic pH values. This novel approach to obtain enzyme loaded, protein resistant nanogel particles is an important alternative for traditional PEGylation strategies and may find broad application in nanomedicine and polymer therapeutics.

Section snippets

Preparation of dPG7.7 functionalized with 10 p-PBDMA units (dPG7.7-10- p-PBDMA)

dPG7.7 (1 g, 0.13 mmol) and p-PBDMA (250 mg, 1.3 mmol) were dissolved in NMP (4 mL) and anhydrous PTSA (22 mg, 0.13 mmol) was added. The reaction was heated to 120 °C for 3 h and the condensed methanol was removed from the reaction equilibrium by cryo-destillation. After cooling down to room temperature (RT) the reaction was quenched by the addition of aqueous ammonia (1 mL). NMP was evaporated by cryo-destillation and the residue was redissolved in basified water (0.05 wt.% aqueous ammonia). The solution

Nanogel preparation by inverse nanoprecipitation and in situ gelation

When biomacromolecules are encapsulated into nanogels in situ to network formation, mildest reaction conditions are required. The nanoprecipitation technology, which has been successfully applied for several years, is based on the injection of highly diluted polymer solutions into polymer non-solvents. This technique is surfactant free and works without high energy input such as ultrasonication and is therefore an excellent candidate for the encapsulation of labile enzymes.

To our knowledge,

Conclusion

We have developed a new approach to generate dendritic polyglycerol nanogels by surfactant free, inverse nanoprecipitation. Size defined nanogels were obtained, while diameters were freely adjustable between 100 and 1000 nm. Applying mild encapsulation conditions we were able to encapsulate the enzyme asparaginase with an efficacy of almost 100%. After degradation triggered release in acidic environments no structural changes of the released cargo were observed and full enzyme activity was

Acknowledgements

We thank Aileen Justies and Dr. Maximilian Zieringer for help with fluorescence and optical microscopy correspondingly. We also thank Cathleen Schlesener for size exclusion measurements. Dr. Pamela Winchester is thanked for carefully proofreading this manuscript. This work was supported by the Helmholtz Virtuelles Institut/Helmholtz-Zentrum and the Focus Area Nanoscale of the Free University Berlin.

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