Nanocomposite formulation for a sustained release of free drug and drug-loaded responsive nanoparticles: an approach for a local therapy of glioblastoma multiforme

Abstract Malignant gliomas are a type of primary brain tumour that originates in glial cells. Among them, glioblastoma multiforme (GBM) is the most common and the most aggressive brain tumour in adults, classified as grade IV by the World Health Organization. The standard care for GBM, known as the Stupp protocol includes surgical resection followed by oral chemotherapy with temozolomide (TMZ). This treatment option provides a median survival prognosis of only 16–18 months to patients mainly due to tumour recurrence. Therefore, enhanced treatment options are urgently needed for this disease. Here we show the development, characterization, and in vitro and in vivo evaluation of a new composite material for local therapy of GBM post-surgery. We developed responsive nanoparticles that were loaded with paclitaxel (PTX), and that showed penetration in 3D spheroids and cell internalization. These nanoparticles were found to be cytotoxic in 2D (U-87 cells) and 3D (U-87 spheroids) models of GBM. The incorporation of these nanoparticles into a hydrogel facilitates their sustained release in time. Moreover, the formulation of this hydrogel containing PTX-loaded responsive nanoparticles and free TMZ was able to delay tumour recurrence in vivo after resection surgery. Therefore, our formulation represents a promising approach to develop combined local therapies against GBM using injectable hydrogels containing nanoparticles

Monoconjugation of Human Amylin with Methylpolyethyleneglycol

<div><p>Amylin is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life <i>in vivo</i> compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF–7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.</p></div

Amylin self-assembly and interaction with co-receptors.

<p>Free murine amylin or PEGylated human amylin were assayed for binding with (<b>A</b>) murine amylin, (<b>B</b>) CTR–1 and (<b>C</b>) RAMP–3 by fluorescence anisotropy of fluorescein labeled molecular partners. Assays were conducted in PBS, pH 7.4, 25°C, in the presence of 50 nM FITC-labelled proteins (amylin, CTR–1 or RAMP–3). Ex 480 nm, Em 520 nm, filter 515 nm. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Amylin and PEG structures.

<p><b>a)</b> Aminoacid sequence of the human and murine amylin. In bold are the different aminoacid. Notice the similarity in sequence from aminoacid 1 to 17. The N-terminus is the Lys1, which comprises the α- and ε-aminogroup, the two unique primary aminogroups in amylin targeted for PEGylation. <b>b)</b> Chemical structure of the methoxyl PEG N-hydroxylsuccinimide (NHS) carbonate. <b>c)</b> Structure of the human amylin. Human amylin (from NMR structure, PDB ID 2KB8) is represented in ribbons colored from aminoacid 1 (blue) to 37 (red). Notice the two aminogroups at the top left side of the representation.</p

Physical stability of the free and the PEGylated human amylin.

<p>Human amylin was subjected to aggregation at 25°C and monitored for fibril formation by ThT fluorescence <b>(a)</b> for up to 12h at varying pH (closed symbols, free non-conjugated human amylin; open symbols, PEGylated human amylin) and <b>(b)</b> for up to 7 days in PBS pH 7.4, and the products of these aggregation kinetic isotherms were further evaluated by transmission electron microscopy (TEM) as follow: <b>(c)</b> free human amylin; <b>(d)</b> PEGylated human amylin; <b>(e)</b> free PEG. Scale bar = 200 nm. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Pharmacologic evaluation of PEGylated human amylin.

<p><b>a)</b> Pharmacokinetics. Non-conjugated and PEGylated human amylin were injected to two separated groups of swiss male mice by subcutaneous administration and the decay in plasma concentration was evaluated. Continuous lines are best fitting of single-exponential decay function to data. <b>b)</b> Modulation of glucagon. Swiss male mice were administered with 10 μg PEGylated human amylin and the serum glucagon was monitored over time. * P<0.05. <b>c)</b> Modulation of glycemia. Swiss male mice (8 weeks old; fasting for 6 h before intervention and throughout the experiment) received by subcutaneous injection regular insulin (0.3 IU/kg body weight) alone (closed circles; n = 5) or in combination with free murine amylin (open circles; 400 μg/kg body weight; n = 5) or PEGylated human amylin (closed inverted triangles; 400 μg/kg body weight, expressed as peptide fraction; n = 5) and glycemia was monitored in the tail tip of free, conscientious mice. The activity of PEGylated human amylin showed significant difference from control (Insulin-R): * P<0.05, **P<0.01. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Amylin-stimulated generation of cAMP in MCF–7 cells.

<p>Non-conjugated or PEGylated amylin were assayed for activity in cell by means of stimulation of production of cAMP in MCF–7 cell line. Lines represent best adjust of experimental data with logistic function. Human amylin EC50 = 35.2 ± 7.5 nM, PEGylated human amylin EC50 = 30.8 ± 6.7 nM. The raw data can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138803#pone.0138803.s005" target="_blank">S2 File</a></b>.</p

Purification and characterization of monoPEGylated human amylin.

<p><b>a)</b> MonoPEGylated human amylin was synthesized in organic solvent by conjugation human amylin (5 mg/mL) with mPEGsc5k (5:1 molar excess) and purified by C18-RP-HPLC with a 30–70% CH₃CN gradient (in the presence of 0.1% TFA) up to 20 min. <b>b)</b> MALDI-ToF-MS of the fractions from the purification step showing the mono PEGylated human amylin corresponding to the peak comprising the elution time 11 min–13 min. The panel is rotated in order to align the PAGE-like display of the mass spectra with the respective original fractions in the chromatogram (upper panel, A). <b>c)</b> Trypsin-digestion of PEGylated human amylin. Purified monoPEGylated human amylin was subjected to trypsin digestion and submitted to MALDI-ToF-MS for identification of the products. The 2748 m/z ion coincides with the expected monoisotopic mass for the sodium adduct of the non-modified (non-PEGylated) human amylin 12–37 fragment (amylin 12–37, amide at C-terminus; monoisotopic mass 2,725.3 Da)</p