INVITED ARTICLE

Self-assembled chitosan-alginate polyplex nanoparticlescontaining temoporfin

Ingrid Brezaniova1,2 & Jiri Trousil3 & Zulfiya Cernochova3 & Vladimir Kral1,2 &

Martin Hruby3 & Petr Stepanek3& Miroslav Slouf3

Received: 11 October 2016 /Revised: 13 November 2016 /Accepted: 16 November 2016# Springer-Verlag Berlin Heidelberg 2017

Abstract The aim of this study was to develop biocompatiblepolyplex nanoparticles with physicochemical properties suit-able for the delivery of photosensitizer temoporfin. We pre-pared, characterized, and compared the two types of polyplexnanoformulations consisting of sodium alginate in combina-tion with chitosan polymer or chitosan oligomer lactate. Weobtained the polyplex system by multiple electrostatic interac-tions between cationic chitosan and anionic alginate and iden-tified key process parameters. Particle size distribution,dispersity, and zeta potential were determined by dynamiclight scattering (DLS), and the diameter and the morphologyof the individual particles were visualized by a transmissionelectronmicroscopy (TEM). It was found that size distributionof the polyplex nanoparticles depends on the concentrations ofchitosan and alginate stock solutions and the order and ratio ofaddition of stock solutions as well as on the pH of the resultingmixture. It appears that the nanoparticles are homogeneous,although micrographs indicate some (vague, indistinct) core-shell structure. The nanoparticles are stable at pH 7.4 (pH ofblood plasma) and show only very little drug leak in experi-ment modeling conditions of blood pool transport to targettissues.

Keywords Chitosan . Sodium alginate . Temoporfin . Drugdelivery system . Photodynamic therapy

AbbreviationsAlg Sodium alginateDH Hydrodynamic diameter (nm)DL Drug loading (%)DLS Dynamic light scatteringEE Entrapment efficiency (%)Chit Chitosan polymerChitOL Chitosan oligomer lactateNPs NanoparticlesPDI DispersityPDT Photodynamic therapyPNPs Polyplex nanoparticlesPS PhotosensitizerSD Standard deviationTEM Transmission electron microscopyT-PNPs Temoporfin-loaded polyplex nanoparticlesUV-VIS Ultraviolet-visible spectroscopyZP Zeta potential (mV)

Introduction

Photodynamic therapy (PDT) is known as a promising thera-peutic modality for the treatment of tumors and non-oncological diseases. The procedure involves the administra-tion of a photosensitizer (PS) followed by local irradiation inthe presence of oxygenwith visible light of a wavelength (e.g.,500–800 nm) appropriate to the absorption spectrum of theparticular PS [1].

Several photosensitizers have been developed for PDT.Tetrapyrrole structures such as porphyrins, chlorins,bacteriochlorins, and phthalocyanines with appropriatefunctionalization have been widely investigated, and severalcompounds have received clinical approval. Other syntheticdye classes tested as PSs involve, e.g., phenothiazinium,

* Martin Hrubymhruby@centrum.cz

1 Faculty of Chemical Engineering, University of Chemistry andTechnology Prague, Technicka 5, 166 28 Prague 6, Czech Republic

2 BIOCEV, Prumyslova 595, 252 50 Vestec, Czech Republic3 Institute of Macromolecular Chemistry, Academy of Sciences of the

Czech Republic, Heyrovsky sq. 2, 162 06 Prague 6, Czech Republic

Colloid Polym SciDOI 10.1007/s00396-016-3992-6

squaraine, boron-dipyrromethene, transition metal complexes,or natural products such as hypericin, riboflavin, andcurcumin [2]. Several authors discussed the development ofsecond generation photosensitizer temoporfin (5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin) and its clinical experiencein a recent review [3–5]. Temoporfin as a hydrophobic por-phyrin derivative has been officially approved in Europe in theyear 2001 for use in clinical treatments, and it is marketedunder the brand name Foscan® for PDT of squamous cellcarcinoma of the head and neck [2] and also for the treatmentof lung, brain, skin, bile duct, and prostate cancer [5].Temoporfin is after intravenous administration adsorbed ontoblood proteins and subsequently translocated into lipoproteins[6]. After internalization into cells, it is localized mainly inmitochondria [7], which cause its extraordinary efficiencycompared to other photosensitizers.

The hydrophobic photosensitizer temoporfin produces itssalt forms just at extreme pH levels, what is not compatiblewith the biological administration. The acid-base properties oftemoporfin were analyzed by Bonnett et al. [8, 9].Spectrophotometric titration of the photosensitizer in amethanol/buffer mixture provided pK3 and pK4 values of3.45 and 1.45, respectively, while the phenolic groups exhib-ited a pKa of 10 [4].

In general, PSs for PDT are most commonly delivered topatients or experimental animals topically or intravenously.Many PSs are hydrophobic or amphiphilic substances whichpossess poor water solubility, and even in the case of watersoluble PSs, there is poor accumulation selectivity at tumorsites. Sometimes, the photosensitizers are administered in al-coholic solution (in ethanol as solvent and propylene glycol ascosolvent) [10]. The alcohol content can induce pain duringadministration. Formulating hydrophobic PSs using lipidic(e.g., Cremophor®) or organic solvent (e.g., propylene glycol)excipients is reasonable for topical or local administration, butcan cause unpredictable biodistribution, toxicity, and hyper-sensitivity if administrated intravenously [11]. To date, varietyof carrier systems based on highly biocompatible materialssuch as liposomes [12–14], micelles [15–18], and polymericNPs [19–21] have been investigated for their potential appli-cation in PS delivery to make the PDT as tumor-selective aspossible.

Natural biodegradable polymers have attracted more atten-tion in formulating PS-delivery nanoparticles due to their out-standing merits such as nontoxicity, biocompatibility, biode-gradability, potential for a multitude of chemical modifica-tions, and low cost [22]. Alginate and chitosan are very prom-ising and have been widely exploited in pharmaceutical indus-try [23]. The ability of some polysaccharides to respond to apH change represents an interesting way to direct delivery ofthe drug to certain tissues or cellular compartments and alsoinfluences the degradation rate of polysaccharides, e.g., ofalginate [24] or chitosan [25], although the degradation of

polysaccharides usually involves several mechanisms includ-ing enzymatic ones [26].

Alginate (Alg) is a water soluble linear polysaccharideextracted from brown seaweed and is composed of alternat-ing blocks of 1 → 4 linked α-L-guluronic and β-D-mannuronic acid residues. The Alg is mucoadhesive, bio-compatible, and biodegradable (naturally by a class of en-zymes called alginate lyases or alginate depolymerases).Easy-gelling property can be used to produce a pre-gelconsisting of very small aggregates of gel-particles, follow-ed by the addition of an aqueous polycationic solution toform a polyelectrolyte complex [23].

Chitosan as cationic polymer is biocompatible, biodegrad-able, and nontoxic in the application of per oral delivery ofdrugs. It is a linear polysaccharide consisting of repeatingunits of D-glucosamine and N-acetyl-D-glucosamine [27]with various degree of deacetylation. Molecular weight anddegree of deacetylation are the main factors affecting the par-ticle size and particle formation and aggregation. Chitosan ispredominantly degraded by enzymes such as lysozyme orglycosidases produced by bacteria in colon [26].

Shieh MJ et al. (2010) [28] prepared the poly(2-ethyl-2-oxazoline)-b-PLA diblock copolymer micelles which werethen used to incorporate temoporfin. The temoporfin-loadedmicelles exhibited efficient PDT effect in vivo, and the thera-peutic efficacy was similar to free drug. However, temoporfin-loaded micelles exhibited less skin phototoxicity than freedrug. Rojnik et al. (2012) [19] prepared temoporfin-loadedPEO-PLGA core-shell NPs. Their in vivo activity wasassessed on athymic nude-Foxn1 mice, and it exhibits thera-peutically favorable tissue distribution. Dark cytotoxicity oftemoporfin delivered by NPs was less than that of freetemoporfin in standard solution (Foscan®), whereas therapeu-tically important phototoxicity was not reduced. Feng et al.(2014) [9] prepared pH-responsive coacervate chitosan-alginate microcapsules for oral administration of doxorubicin(DOX) with excellent low pH tolerance (stability of the algi-nate core in the stomach) and rapid drug release in the smallintestine.

In the present study, we prepared and characterized the twotypes of nanocarriers for second generation photosensitizertemoporfin composed of chitosan polymer or chitosan oligo-mer lactate, respectively, with sodium alginate. To the best ofour knowledge, the chitosan-alginate polyplexes were not de-scribed as a PDT drug carrier, and there was also no studycritically comparing the effect of molecular weight of chitosanon the properties of polyplexes prepared thereof. Polyplexnanoparticles are formed by multiple electrostatic interactionsand hydrogen bond between cationic chitosan polymer andoligomer, respectively, and anionic alginate. The nanoparti-cles showed suitable properties (such as size) for potentialuse PDT after topical, intraperitoneal, or lymph nodemetastases-directed PDT.

Colloid Polym Sci

Materials and methods

Materials

Chitosan [product number: 448869, molecular weight50,000–190,000 Da (based on viscosity), 75–85%deacetylated], chitosan oligosaccharide lactate [product num-ber: 523682, molecular weight 4000–6000 (based on viscos-ity), 90% deacetylated], sodium alginate [product number:180947, molecular weight 40,000–150,000 Da (based on vis-cosity)], sodium hydroxide, sodium chloride, anhydrous mag-nesium sulfate, acetic acid, 1-octanol, acetonitrile, and meth-anol were obtained from Sigma-Aldrich Ltd. (Prague,Czech Republic). Temoporfin has been synthesized accordingto ref. [29], and purity of the synthesized temoporfin was> 98% (HPLC), 1H NMR (600 MHz, DMSO-d6) δ = −1.64(s, 2H), 4.14 (s, 4H), 7.08 (m, 2H), 7.15 (m, 2H), 7.26 (m,2H), 7.29 (m, 2H), 7.45 (m, 2H), 7.50 (m, 6H), 8.25 (d, 2H),8.40 (s, 2H), 8.65 (d, 2H), 9.79 (s, 2H), 9.84 (s, 2H); MS-ESI+

681 M + H+.

Preparation of stock solutions

We prepared the two types of polyplex nanoformulations, typeA (from chitosan polymer) and type B (from chitosan oligo-mer lactate). The composition of stock solutions is shown inScheme 1.

Investigation of the conditions for the formationof temoporfin-loaded polyplex nanoparticles

Formation of polyplex nanoparticles dependingon chitosan-alginate ratio and pH

Formation of PNPs was studied by mixing different volumeratios of cationic chitosan and anionic alginate stock solutionsat different pH as shown in Tables 2 and 3. The chitosan:

alginate monomeric unit molar ratio was calculated from themass concentration, known monomeric unit, and assumptionthat the sugar unit carries a one positive charge of chitosan andone negative charge of alginate.

Preliminary experiments were done in order to determineratios leading to nanoparticles formation. For this purpose,variable amounts of Chit/ChitOL stock solutions were addeddropwise above the surface of variable amounts of Alg stocksolution (pH ∼12) under magnetic stirring at room tempera-ture. The resulting mixture was left stirring for 1 day at roomtemperature. The samples were visually analyzed and threedifferent results were identified: clear solution not containingnanoparticles, opalescent nanoparticle solution, and macro-scopic aggregates/precipitate, respectively. Opalescent solu-tions containing nanoparticles were then further investigated.

The pH value of prepared bulk PNPs was determined witha pH meter (FE20-FiveEasy™ ; Met t le r Toledo,Czech Republic). The pH values were adjusted with sodiumhydroxide (11%) and with acetic acid (1%). Consequently, thepH values of the selected Chit-Alg nanoparticles were set to∼5 (pH in endosomes after internalization into target cells) and7.4 (pH of blood plasma), respectively. To obtain equilibrium,each tested formulation was left undisturbed for a few minutesafter thorough stirring. Prior to analysis, all the prepared sam-ples were centrifuged at 4000 rpm for 3 min (MiniSpin™Microcentrifuges; Eppendorf™, Germany) to remove eventu-al large aggregates, and 1 mL of supernatant was removed andfurther analyzed.

Preparation of temoporfin-loaded polyplex nanoparticles

As schematically presented in Fig. 1, preparations oftemoporfin-loaded polyplex nanoformulations were obtainedby multiple electrostatic interactions between cationic chito-san and anionic alginate with non-covalently encapsulatedtemoporfin.

Briefly, 1 mL of temoporfin solution (1 mg/1 mL in meth-anol) was added dropwise above the surface of 7.5 mL of Alg

Scheme 1 The stock solutionsused to produce temoporfin-loaded polyplexnanoformulations type A (fromchitosan polymer) and type B(from chitosan oligomer lactate)

Colloid Polym Sci

stock solution (pH ∼12) under magnetic stirring at room tem-perature. After that, 2.5 mL of chitosan (Chit or ChitOL, re-spectively) stock solution (pH ∼5) was added dropwise abovethe surface of temoporfin-containing Alg solution under mag-netic stirring. The particles were left stirring for 1 day. Prior toanalysis, samples were centrifuged (3 min/4000 rpm), and1 mL of supernatant was removed and further analyzed.

Dynamic light scattering measurements

Dynamic light scattering (DLS) measures diffusion in nano-particle dispersions. Particle size (DH), dispersity (PDI), andzeta potential (ZP) of nanoparticles were determined (at25 °C) with a Zetasizer (Zen 3600; Malvern InstrumentsLtd.) instrument, using a He–Ne ion laser (λ = 633 nm) at ascattering angle of 173°. All samples were ten times dilutedwith ultrapure water to reach a suitable concentration beforemeasurement and to enable its fully efficient ionic colloidalstabilization. The mean average of peak positions in theintensity-weighted distribution of sizes was used to representdata. Zeta potential values were calculated by theSmoluchowski’s approximation, converting electrophoreticmobility to zeta potential values (mV). The Zetasizer software(version 9.01; Malvern Instruments Ltd.) generated measure-ment data. The measurements (six readings for each assay)

were performed in a cell. The assays were performed in trip-licate, so the results are given as the average ± standard devi-ation of the 18 values obtained.

Transmission electron microscopy (TEM)

The samples T-PNPs type A and B, respectively, were visual-ized with a TEM microscope (Tecnai G2 Spirit Twin 12; FEI,Czech Republic). For each sample, 2 μL of the suspensionwas dropped onto a holey carbon-coated TEM grid; the excesssolution was sucked off after 1 min by touching the bottom ofthe grid with a thin strip of filter paper in order to removepossible soluble impurities. The particles were left to drycompletely at room temperature and observed in the TEMmicroscope (bright field imaging, accelerating voltage120 kV).

UV-VIS spectroscopy

Determination of photosensitive temoporfin was carried outon a flexible monochromator-based multi-mode microplatereader (Synergy™ H1 Hybrid Reader; Biotek, USA) using amicroplate (Nunc™ 96-Well; Thermo Scientific, USA). Allsamples were diluted to reach a suitable concentration beforemeasurement. The measurements were carried out at 25 °C.

Fig. 1 Preparation of the temoporfin-loaded polyplex nanoparticles. Nanoformulations with non-covalently encapsulated temoporfin were obtained bymultiple electrostatic interactions and hydrogen bonding between cationic chitosan and anionic alginate

Colloid Polym Sci

Temoporfin has an absorption peak in the red region atλ = 652 nm with high molar extinction coefficient(ε ∼2.9 × 104 M−1 cm−1), and it has another absorption band inthe blue region centered at 417 nmwith even much higher molarextinction coefficient (Soret band, ε ∼2.3 × 105 M−1 cm−1) [30].

Drug loading and entrapment efficiency determination

Drug incorporation efficiency was expressed both as drugcontent, in the literature also referred to as drug loading (DL,%) and entrapment efficiency (EE,%), represented by Eqs. (1)and (2), respectively:

DL ¼ Mass of temoporfin in nanoparticles

Mass of nanoparticles with temoportin100% ð1Þ

EE ¼ Mass of temoporfin in nanoparticles

Mass of temoporfin used for the formulation100% ð2Þ

The mass of drug (temoporfin) in nanoparticles was calcu-lated as a difference between the mass of non-encapsulateddrug and the mass of drug used for the nanoformulation (i.e.,total mass of temoporfin in the formulation).

Mass of non-encapsulated drug that was in equilibriumwith the NPs was determined in a filtrate that was obtainedby ultrafiltration (10 min/7000 rpm, 25 °C) using an AmiconUltra centrifugal filter units (Ultra-15, NMWL of 100 kDa;Millipore, Ireland).

Total mass of temoporfin in the investigated formulationswas determined by a procedure based on QuEChERS method[31]. Briefly, 5 mL of the T-PNP formulation (type A or B,respectively) and 5 mL of acetonitrile were pipetted to a cen-trifugation tube, and this mixture was shaken vigorously byhand for 5 min. After that, 500 mg of each sodium chlorideand anhydrous magnesium sulfate was added, and the mixturewas shaken vigorously by hand for 5 min. Temoporfin wasdetermined in the upper acetonitrile phase after centrifugation(5 min/7000 rpm; 25 °C). Quantification of analyte temoporfinwas performed by UV-VIS spectroscopy by a standard additionmethod. For our purposes, the absorbance was measured atλ = 417 nm (ε ∼2.3 × 105 M−1 cm−1).

In vitro release of temoporfin—modeling delivery systemstability during transport in bloodstream

The in vitro release of temoporfin was evaluated using a 1-octanol extraction method [32]. Five milliliters of each 1-octanol and the studied formulation of temoporfin-loadednanoparticles (pH 7.4) were pipetted to a centrifugation tube.Drug release experiments were carried out using a temperature-controlled shaker at 37 °C and 100 rpm. Release of temoporfinwas observed during the extraction as an increase of measuredabsorbance. For this purpose, an aliquot (300 μL) of upper 1-octanol phase was collected, and the content of temoporfin in it

was determined by UV-VIS spectrophotometry at λ = 417 nm(ε ∼2.3 × 105 M−1 cm−1).

Statistical analysis

Chemical, physicochemical, and drug release experimentswere done in three replications. Data are expressed as arith-metic mean values with the corresponding standard deviations(± SD). Statistical analysis of data was performed by Originprogram (version 9.0, OriginLab Co., MA, USA).

Results and discussion

Preparation and physicochemical characterizationof empty and temoporfin-loaded polyplex nanoparticles

Among different methods available for the preparation of NPs[23, 33, 34], electrostatic complexation and hydrogen bond-ing, generally referred to as ionotropic gelation method, wasused in this study for the preparation of chitosan-alginate-based NPs. Nanoparticles were obtained spontaneously undervery mild conditions.

A set of experiments was carried out in a systematic way tofind the optimal conditions for the development of two typesof polyplex nanoformulations, typeA (from chitosan polymer,Chit) and type B (from chitosan oligomer lactate, ChitOL).These two formulations differ in molecular weight of chitosancomponent. Preliminary experiments were done in order todetermine the production zone of the required nanoparticleformation, namely, variable amounts of Chit, ChitOL, andAlg stock solutions for polyplex nanoparticle formation weretested. Selected chitosan (Chit or ChitOL): alginate monomer-ic unit molar ratios tested are shown in Tables 1 and 2. Weused the approximate value for the density of the particles 1 g/mL as usual for such polymer particles with high content ofwater.

Chitosan and alginate are weak polyelectrolytes, meaningthat their dissociation degree depends strongly on the solutionpH. As it was previously reported, when the pH of Alg solutionis below or close to the pKa value (pH ∼3.4–4.4), a drasticdecrease in the ionization degree of Alg macromolecules isobserved leading to a lack of sufficient charge. Chitosan is onlysoluble in acidic solutions, where the pH value is sufficientlybelow its pKa (6.2–7.0) [35, 36]. In order to ensure theirpolycation and polyanion behavior, respectively, the pH valuesof the Chit-alginate-based NPs and ChitOL-alginate-based NPswere set at ∼5 and 7.4, respectively (Tables 1 and 2).

The DLS technique was used to determine ensemble sizeaverages of dispersed particles. It was found that the size dis-tribution of the nanosystem depends on the concentration ofchitosan and alginate solutions and their ratio as well as on thepH of their mixture. Interestingly, size of higher molecular

Colloid Polym Sci

weight chitosan-based Chit-Alg NPs is more sensitive to theChitOL: Alg monomeric unit molar ratio and pH used com-pared to the size of the low molecular weight chitosanoligomer-based nanoparticles, which is logical because theoligomer behaves more like multivalent, yet relatively smallcation, while the higher molecular weight chitosan is a truepolycation. As shown in Figs. 2 and 3, the most appropriateparticle sizes were achieved when the Chit: Alg and ChitOL:Alg monomeric unit molar ratio, respectively, was 1:3 (i.e.,monomeric unit molar fraction of chitosan in particles com-position was 0.25), and when pH value was set at the pH ofblood plasma (pH 7.4). In this case, the particle size (DH) ofChit-Alg NPs was ∼300 nm and DH of ChitOL-Alg NP was∼350 nm. These experiment settings were used for formationof two types of polyplex nanoformulations, type A (from chi-tosan polymer) and type B (from chitosan oligomer lactate).

The excess of alginate is necessary for efficient electrostaticstabilization of the NPs against aggregation due to efficientnegative surface charge (expressed as ζ-potential, ZP, see be-low). The negative overall charge of the NPs is also advanta-geous compared to positive charge to avoid unwanted nonspe-cific coulombic interaction with body tissues, membranes, andproteins mostly having negative overall charge.

The excess of alginate as weak polyacid is also causing thepH dependence of size; the electrostatic stabilization is moreefficient at pH 7.4 where the polyacid is completely ionized,while at pH 5.0, the stabilization is less efficient and the par-ticles are larger.

In order to determine the nanoparticle size distribution(DH), PDI and ZP of temoporfin-loaded polyplexnanoformulations, type A (from chitosan polymer) and type

B (from chitosan oligomer lactate), respectively, we used DLStechnique. From the results obtained (Table 3), DH of T-PNPstype Awas ∼500 nm and DH of T-PNPs type B was ∼700 nm.

Fig. 2 The particle size (DH) of chitosan polymer-alginate nanoparticlesin dependence on their monomeric unit molar fraction of chitosanmeasured by dynamic light scattering as well as on the pH (5, 7.4) ofthe mixture. The composition of stock solutions: chitosan polymer(36.9 mg/30 ml) in 2% acetic acid and sodium alginate (30.0 mg/30 ml)in distilled water

Table 2 Chitosan oligomer lactate: Alginate monomeric unit molarratio in selected nanoparticle polymeric systems at pH ∼5 and ∼7.4.The composition of stock solutions: chitosan oligomer lactate (30.0 mg/30 ml) in 2% acetic acid and sodium alginate (52.4 mg/30 ml) in distilledwater

pH of ChitOL-AlgNPs solution

ChitOL: Algmonomericunit molar ratio

pH ∼5 1.5:1 1:1 1:1.5 1:2 1:2.5 1:3

pH ∼7.4 1:1 1:1.5 1:2 1:2.5 1:3 –

Table 1 Chitosan polymer: Alginate monomeric unit molar ratio inselected nanoparticle polymeric systems at pH ∼5 and ∼7.4. Thecomposition of stock solutions: chitosan polymer (36.9 mg/30 ml) in2% acetic acid and sodium alginate (30.0 mg/30 ml) in distilled water

pH of Chit-AlgNPs solution

Chit: Alg monomeric unit molar ratio

pH ∼5 2:1 1.25:1 1:1.5 1:2

pH ∼7.4 2:1 1:2 1:2.5 1:3

Fig. 3 The particle size (DH) of chitosan oligomer lactate-alginatenanoparticles in dependence on their monomeric unit molar fraction ofchitosan (measured by dynamic light scattering) as well as on the pH (5,7.4) of the mixture. The composition of stock solutions: chitosanoligomer lactate (30.0 mg/30 ml) in 2% acetic acid and sodium alginate(52.4 mg/30 ml) in distilled water

Colloid Polym Sci

The zeta potential of T-PNPs type A was ∼−33 mV and of T-PNPs type B was ∼−45 mV, which corresponds with efficientelectrostatic stabilization of the particles in solution. Generally,values more electronegative than −30 mV represent sufficientmutual repulsion to result in stability (i.e., no agglomeration)[37]. The PDI of T-PNPs (type A or B) nanodispersion was∼0.4. Size of the type B nanoparticles (from chitosan oligomerlactate, ChitOL) also changes more by temoporfin incorpora-tion than size of the polymeric chitosan-based ones; this makessense if we imagine that longer polymers generally form morestable interpolyelectrolyte complexes.

The obtained results show an influence of chitosan molec-ular weight on nanoparticles size and zeta potential. Overall,the use of chitosan polymer of lower molecular weight (Chit)led to the formation of smaller particles with more negativeZP. This may stem from the ability of chitosan of lower mo-lecular weight to diffuse more promptly in the alginate gelmatrix to form smaller nanoparticles. Whereas, on the con-trary, chitosan oligomers of higher molecular weight or vis-cosity may bind to the surface of such matrices, forming anouter membrane and leading to increase in particle size anddecrease in ZP in consequence of alginate excess.

Transmission electron microscopy (TEM)

The TEM studies were conducted to investigate the size andshape of dried individual temoporfin-loaded polyplex

nanoparticles type A (with Chit) or B (with ChitOL) (seeFig. 5), which were deposited on electron transparent carbonfilm. The particles appear as black spots on gray background.It appears that samples are relatively homogeneous, althoughmicrographs indicate some (vague, indistinct) core-shell struc-ture. The diameter of temoporfin-loaded polyplex nanoparti-cles, measured in TEM micrographs, was approximately100 nm (type A nanoparticles; Fig. 4a) and 150 nm (type Bnanoparticles; Fig. 4b). Higher size of type B nanoparticleswas in agreement with DLS experiments (Table 3), but thenanoparticle diameters determined from TEM were systemat-ically smaller in comparison with DLS for both type A andtype B nanoparticles. There is often a discrepancy betweenTEM and DLS measurements that is attributed to factors as-sociated with the high vacuum conditions of TEM and thehydrodynamic, electrokinetic effects influencing DLS mea-surements. TEM provides the size distribution of dehydratedpolyplex particles, while DLS measures the apparent size of aparticle, including hydration layers that form around hydro-philic particles such as those composed of chitosan-alginate[38, 39].

Drug loading and entrapment efficiencydetermination

Temoporfin incorporation efficiency was expressed as com-monly used values of drug loading (DL) and entrapment

Table 3 Particle size (DH), dispersity (PDI), and zeta potential (ZP) of temoporfin-loaded polyplex nanoformulations type A (from chitosan polymer)and type B (from chitosan oligomer lactate), respectively, measured by dynamic light scattering, (mean ± SD, n = 3). Monomeric unit molar ratio ofchitosan polymer to alginate or chitosan oligomer lactate to alginate, respectively, was 1:3 at pH ∼7.4

Formulation Size (DH, nm) ± SD PDI ± SD ZP (mV) ± SD

Temoporfin-loaded polyplex NPs—type A(i.e., with chitosan polymer)

498 ± 39 0.444 ± 0.012 −32.64 ± 0.88

Temoporfin-loaded polyplex NPs—type B(i.e., with chitosan oligomer lactate)

728 ± 47 0.376 ± 0.043 −45.20 ± 0.22

Fig. 4 TEM micrographs ofdried temoporfin-loaded polyplexnanoparticles. a Temoporfin-loaded chitosan polymer-alginatenanoparticles. b Temoporfin-loaded chitosan oligomer lactate-alginate nanoparticles.Monomeric unit molar ratio ofchitosan polymer to alginate orchitosan oligomer lactate toalginate, respectively, was 1:3 atpH ∼7.4

Colloid Polym Sci

efficiency (EE), that is based on knowledge of the drug con-centration in the nanoparticles as well as in the free (non-encapsulated) form [40, 41]. Determination of these valueswas complicated by the complex character of the studied ma-trix, since polysaccharide nanoparticles are insoluble inorganic solvents.

Thus, another way for the determination of the total amountof temoporfin in the formulations was found. For this purpose,an approach based on the QuEChERS extraction method, thatwas developed primarily for food matrixes with high contentof water, was used [31].

Then, the UV-VIS determination of temoporfin gavevalues of DL and EE. For the T-PNP type A (i.e., with Chit)nanoparticles, the values of EE and DL were 61.8 and 6.3%,respectively. For the T-PNP type B (i.e., with ChitOL) nano-particles, the method provided slightly higher values of EEand DL which were 72.8 and 8.3%, respectively.

In vitro release study

Release of temoporfin from polyplex nanoparticles was eval-uated. For this purpose, 1-octanol extraction method was usedas a highly relevant model of the relative rate of uptake ofnumerous poorly water soluble hydrophobic drugs via mem-brane barriers in organisms [42–44]. Moreover, the 1-octanolmethod was preferred in our work instead of the commonlyused dialysis method, that is referred as a method providingmisleading data by several authors (e.g., [45–47]), becausedialysis data obtained from the nanoformulations is not nec-essarily descriptive of the kinetics of the release fromnanoparticulate systems.

The release of temoporfin was found to be linear (zero-order kinetics) with quantitative release (100%) within14 days. The high coefficients of determination of T-PNP typeA (R2 = 0.9966) and T-PNP type B (R2 = 0.9853) were ob-served (see Fig. 5). The release curves illustrate that the releaseof temoporfin from the aqueous phase into the organic layer isconsiderably slow in accordance with that temoporfin is alipophilic drug with log P of 5.5 [48] and showing superiorstability of temoporfin encapsulation inside the particles,which is necessary to efficiently transport the cargo into thetarget cancerous tissue.

Conclusion

The two types of temoporfin-loaded polyplex nanoparticlesbased on sodium alginate and chitosan as polymer or chitosanoligomer lactate have been successfully prepared and charac-terized. These self-assembling nanoparticles with non-covalently encapsulated temoporfin were obtained via multi-ple electrostatic interaction and hydrogen bonding betweencationic chitosan and anionic alginate. The size of preparednanoparticles was measured by DLS (nanoparticles in solu-tion) and TEM (dried nanoparticles). It was found that the sizedistribution (DLS) of the polyplex nanoparticles depends onthe concentrations of chitosan and alginate stock solutions, theorder and ratio of addition, as well as on the pH of the resultingmixture. The TEM studies indicated that nanoformulations arehomogeneous, although micrographs indicate some (vague,indistinct) core-shell structure. The in vitro release oftemoporfin from polyplex nanoparticles was evaluated by 1-octanol extraction, and linear zero-order time drug release

Fig. 5 Cumulative releasepercentage (%)—time profiles oftemoporfin and its estimation bylinear interpolation (zero-orderkinetics). Time temoporfin releaseprofiles from formulations: T-PNP type A (temoporfin-loadedpolyplex nanoparticles type A(i.e., with chitosan polymer)) andPNP type B (temoporfin-loadedpolyplex nanoparticles type B(i.e., with chitosan oligomerlactate)). Monomeric unit molarratio of chitosan polymer toalginate or chitosan oligomerlactate to alginate, respectively,was 1:3 at pH ∼7.4

Colloid Polym Sci

profiles (100% within 14 days) were observed thus showingsuperior stability of temoporfin encapsulation inside the par-ticles, which is necessary to efficiently transport the cargo intothe target cancerous tissue. These biodegradable and stablenanoparticles at the pH of blood plasma (pH 7.4) are suitablefor photodynamic therapy after topical application or for in-traperitoneal PDT. Furthermore, they might be used as thenanocarriers, which can deliver temoporfin into lymphatic tis-sues, including lymph node metastases, and then release thedrug inside these lesions.

Acknowledgments This work was supported by the Ministry ofEducation, Youth and Sports of the Czech Republic within the LQ1604National Sustainability Program II (Project BIOCEV-FAR) and by theproject BBIOCEV^ (CZ.1.05/1.1.00/02.0109). This work was supportedby the Ministry of Education, Youth and Sports of the Czech Republicgrant no. LH14008 (Contact II). The author is grateful for the supports bythe IGA University of Chemistry and Technology, Prague, no. A1_FCHI_2016_003. The authors from Institute of MacromolecularChemistry AS CR acknowledge financial support from Czech ScienceFoundation (grant no. 16-02870S) and from Ministry of Health of theCzech Republic (grant no. 15-25781a). Electron microscopy at theInstitute of Macromolecular Chemistry was supported by projectPOLYMAT LO1507 (Ministry of Education, Youth and Sports of theCR, program NPU I).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest.

References

1. Lucky SS, Soo KC, Zhang Y (2015) Nanoparticles in photodynam-ic therapy. Chem Rev 115:1990–2042. doi:10.1021/cr5004198

2. Abrahamse H, Hamblin MR (2016) New photosensitizers for pho-todynamic therapy. Biochem J 4:347–364. doi:10.1042/BJ20150942

3. SengeMO (2012)MTHPC—a drug on its way from second to thirdgeneration photosensitizer? Photodiagn Photodyn Ther 9:170–179.doi:10.1016/j.pdpdt.2011.10.001

4. Senge MO, Brandt JC (2011) Temoporfin (Foscan®, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin)—a second-generation photosensi-tizer. Photochem Photobiol 87:1240–1296. doi:10.1111/j.1751-1097.2011.00986.x

5. Agostinis P, Berg K, Cengel KA, et al. (2011) Photodynamic ther-apy of cancer: an update. AmCancer Soc 61:250–281. doi:10.3322/caac.20114.Available

6. Sasnouski S, Zorin V, Khludeyev I, et al. (2005) Investigation ofFoscan® interactions with plasma proteins. Biochim Biophys Acta-Gen Subj 1725:394–402. doi:10.1016/j.bbagen.2005.06.014

7. François A,Marchal S, Guillemin F, Bezdetnaya L (2011)mTHPC-based photodynamic therapy induction of autophagy and apoptosisin cultured cells in relation to mitochondria and endoplasmic retic-ulum stress. Int J Oncol 39:1537–1543. doi:10.3892/ijo.2011.1174

8. Bonnett R, Djelal BD, Nguyen A (2001) Physical and chemicalstudies related to the development of m-THPC (FOSCAN (R))for the photodynamic therapy (PDT) of tumours. J PorphyrinsPhthalocyanines 5:652–661

9. Feng C, Song R, Sun G, et al. (2014) Immobilization of coacervatemicrocapsules in multilayer sodium alginate beads for efficient oralanticancer drug delivery. Biomacromolecules 15:985–996.doi:10.1021/bm401890x

10. Hofman JW, Carstens MG, van Zeeland F, et al. (2008)Photocytotoxicity of mTHPC (temoporfin) loaded polymeric mi-celles mediated by lipase catalyzed degradation. Pharm Res 25:2065–2073. doi:10.1007/s11095-008-9590-7

11. Li L, Huh KM (2014) Polymeric nanocarrier systems for photody-namic therapy. Biomater Res 18:19. doi:10.1186/2055-7124-18-19

12. Derycke ASL, DeWitte PAM (2004) Liposomes for photodynamictherapy. Adv Drug Deliv Rev 56:17–30. doi:10.1016/j.addr.2003.07.014

13. Reshetov V, Lassalle H-P, François A, et al. (2013) Photodynamictherapy with conventional and PEGylated liposomal formulationsof mTHPC (temoporfin): comparison of treatment efficacy and dis-tribution characteristics in vivo. Int J Nanomedicine 8:3817–3831.doi:10.2147/IJN.S51002

14. Dragicevic-Curic N, Fahr A (2012) Liposomes in topical photody-namic therapy. Expert Opin Drug Deliv 9:1015–1032. doi:10.1517/17425247.2012.697894

15. Taillefer J, Brasseur N, van Lier JE, et al. (2001) In-vitro and in-vivo evaluation of pH-responsive polymeric micelles in a photody-namic cancer therapy model. J Pharm Pharmacol 53:155–166.doi:10.1211/0022357011775352

16. Gibot L, Lemelle A, Till U, et al. (2014) Polymeric micelles encap-sulating photosensitizer: structure/photodynamic therapy efficiencyrelation. Biomacromolecules 15:1443–1455. doi:10.1021/bm5000407

17. Van Nostrum CF (2004) Polymeric micelles to deliver photosensi-tizers for photodynamic therapy. Adv Drug Deliv Rev 56:9–16.doi:10.1016/j.addr.2003.07.013

18. Koo H, Lee H, Lee S, et al. (2010) In vivo tumor diagnosis andphotodynamic therapy via tumoral pH-responsive polymeric mi-celles. Chem Commun (Camb) 46:5668–5670. doi:10.1039/c0cc01413c

19. Rojnik M, Kocbek P, Moret F, et al. (2012) In vitro and in vivocharacterization of temoporfin-loaded PEGylated PLGA nanopar-ticles for use in photodynamic therapy. Nanomedicine 7:663–677.doi:10.2217/nnm.11.130

20. Fadel M, Kassab K, Abdel Fadeel D (2010) Zinc phthalocyanine-loaded PLGA biodegradable nanoparticles for photodynamic ther-apy in tumor-bearing mice. Lasers Med Sci 25:283–292.doi:10.1007/s10103-009-0740-x

21. Zeisser-LabouèbeM, Lange N,GurnyR, Delie F (2006) Hypericin-loaded nanoparticles for the photodynamic treatment of ovariancance r. I n t J Pha rm 326 :174–181 . do i : 10 . 1016 / j .ijpharm.2006.07.012

22. Debele TA, Peng S, Tsai HC (2015) Drug carrier for photodynamiccancer therapy. Int J Mol Sci. doi:10.3390/ijms160922094

23. Li P, Dai Y, Zhang J, et al. (2008) Chitosan-alginate nanoparticles asa novel drug delivery system for nifedipine. Int J Biomed Sci 4:221–228

24. Haug A, Larsen B, Smidsrød O (1966) A study of the constitutionof alginic acid by partial acid hydrolysis. Acta Chem Scand 20:183–190. doi:10.3891/acta.chem.scand.20-0183

25. Holme HK, Foros H, Pettersen H, et al. (2001) Thermal depolymer-ization of chitosan chloride. Carbohydr Polym 46:287–294.doi:10.1016/S0144-8617(00)00332-5

26. Guarino V, Caputo T, Altobelli R, Ambrosio L (2015) Degradationproperties and metabolic activity of alginate and chitosan polyelec-trolytes for drug delivery and tissue engineering applications.AIMS Mater Sci 2:497–502. doi:10.3934/matersci.2015.4.497

27. Hejazi R, Amiji M (2003) Chitosan-based gastrointestinal deliverysystems. J Control Release 89:151–165. doi:10.1016/S0168-3659(03)00126-3

Colloid Polym Sci

28. Shieh M-J, Peng C-L, Chiang W-L, et al. (2010) Reduced skinphotosensitivity with meta-tetra(hydroxyphenyl)chlorin-loaded mi-celles based on a poly(2-ethyl-2-oxazoline)-b-poly(d,l-lactide)diblock copolymer in vivo. Mol Pharm 7:1244–1253. doi:10.1021/mp100060v

29. Banfi S, Caruso E, Buccafurni L, et al. (2006) Synthesis, photody-namic activity, and quantitative structure-activity relationshipmodeling. J Med Chem 49:3293–3304

30. Yu Q, Rodriguez EM, Naccache R, et al. (2014) Chemical modifi-cation of temoporfin—a second generation photosensitizer activatedusing upconverting nanoparticles for singlet oxygen generation.Chem Commun (Camb) 50:12150–12153. doi:10.1039/c4cc05867d

31. Anastassiades M, Lehotay SJ (2003) Fast and easy multiresiduemethod employing acetonitrile extraction/partitioning andBdispersive solid-phase extraction^ for the determination of pesti-cide residues in produce. J AOCA Int 86:412–431

32. Trousil J, Panek J, Hruby M, et al. (2014) Self-association of beepropolis: effects on pharmaceutical applications. J Pharm Investig44:15–22. doi:10.1007/s40005-013-0104-1

33. Abdelghany SM, Schmid D, Deacon J, et al. (2013) Enhancedantitumor activity of the photosensitizer meso-tetra(N-methyl-4-pyridyl) porphine tetra tosylate through encapsulation inan t ibody- t a rge t ed ch i to san / a lg ina t e nanopa r t i c l e s .Biomacromolecules 14:302–310. doi:10.1021/bm301858a

34. Jawahar N, Meyyanathan S (2012) Polymeric nanoparticles fordrug delivery and targeting: a comprehensive review. Int J HealAllied Sci 1:217. doi:10.4103/2278-344X.107832

35. RiveraMC, Pinheiro AC, BourbonAI, et al. (2015) Hollow chitosan/alginate nanocapsules for bioactive compound delivery. Int J BiolMacromol 79:95–102. doi:10.1016/j.ijbiomac.2015.03.003

36. Tan ML, Choong PFM, Dass CR (2009) Cancer, chitosan nanopar-ticles and catalytic nucleic acids. J Pharm Pharmacol 61:3–12.doi:10.1211/jpp/61.01.0002

37. Duan J, Wang J, Guo T, Gregory J (2014) Zeta potentials and sizesof aluminum salt precipitates—effect of anions and organics andimplications for coagulation mechanisms. J Water Process Eng 4:224–232. doi:10.1016/j.jwpe.2014.10.008

38. Ito T, Sun L, Bevan MA, Crooks RM (2004) Comparison of nano-particle size and electrophoretic mobility measurements using acarbon-nanotube-based coulter counter, dynamic light scattering,transmission electron microscopy, and phase analysis light scatter-ing. Langmuir 20:6940–6945. doi:10.1021/la049524t

39. Douglas KL, Tabrizian M (2005) Effect of experimental parameterson the formation of alginate-chitosan nanoparticles and evaluationof their potential application as DNA carrier. J Biomater Sci PolymEd 16:43–56. doi:10.1163/1568562052843339

40. Singh R, Lillard JW (2009) Nanoparticle-based targeted drug deliv-ery. Exp Mol Pathol 86:215–223. doi:10.1016/j.yexmp.2008.12.004

41. Joye IJ, McClements DJ (2014) Biopolymer-based nanoparticles andmicroparticles: fabrication, characterization, and application. Curr OpinColloid Interface Sci 19:417–427. doi:10.1016/j.cocis.2014.07.002

42. Bender KI, Lutsevich AN (1988) Dependence of the absorptionthrough the human oral mucosa of antihistaminic (H1) preparationson their physicochemical properties. Farmakol Toksikol 51:75–79

43. Vasiluk L (2006) Development of an in vitro system to access theoral bioavailability of hydrophobic contaminants. Simon FraserUniversity, Burnaby, Canada

44. Waring MJ (2009) Defining optimum lipophilicity and molecularweight ranges for drug candidates-molecular weight dependentlower log D limits based on permeability. Bioorganic Med ChemLett 19:2844–2851. doi:10.1016/j.bmcl.2009.03.109

45. ZambitoY, Pedreschi E,DiColoG (2012) Is dialysis a reliablemethodfor studying drug release from nanoparticulate systems? A case study.Int J Pharm 434:28–34. doi:10.1016/j.ijpharm.2012.05.020

46. Moreno-Bautista G, Tam KC (2011) Evaluation of dialysis mem-brane process for quantifying the in vitro drug-release from colloi-dal drug carriers. Colloids Surfaces A Physicochem Eng Asp 389:299–303. doi:10.1016/j.colsurfa.2011.07.032

47. Modi S, Anderson BD (2013) Determination of drug release kinet-ics from nanoparticles: overcoming pitfalls of the dynamic dialysismethod. Mol Pharm 10:3076–3089. doi:10.1021/mp400154a

48. Mehraban N, Freeman HS (2015) Developments in PDTsensitizersfor increased selectivity and singlet oxygen production. Materials(Basel). doi:10.3390/ma8074421

Ingrid Brezaniova is pursuing her Ph.D. under Professor Vladimír Král at theUniversity of Chemistry and Technology in Prague, Czech Republic. She hasfor many years enjoyed the collaboration with the Institute of MacromolecularChemistry AS CR, v.v.i., Prague (Martin Hrubý and Petr Štěpánek). Sheworked with the Institute of Biochemistry and Experimental Oncology atFirst Faculty of Medicine, Charles University in Prague (Pavel Martásek).Her research focuses on development, preparation and characterization ofnew drug delivery systems for photodynamic anticancer therapy such as (1)thermoresponsive solid lipid nanoparticles; (2) silica-based nanoparticles; and(3) chitosan-alginate polyplex nanoparticles.

Colloid Polym Sci

Jiri Trousil M.Sc. is a Ph.D. student (supervised by Dr. Martin Hrubý) atDepartment of Analytical Chemistry, Faculty of Science, Charles Universityin Prague, Czech Republic. He got both BSc. and MSc. in forensic sciences atUniversity of Chemistry and Technology Prague, Czech Republic. At the sameinstitute, he got second BSc. in chemistry teaching. Currently his research isrealized at the Institute of Macromolecular Chemistry of Academy of Sciencesof the Czech Republic in the department led by professor Petr Štěpánek and itis focused on preparation and analysis of biologically active nanoparticles andits behavior under bio-relevant conditions with accent on infection diseases.

Zulfiya Cernochova has been research fellow at Institute of MacromolecularChemistry AS CR v.v.i., Department of Supramolecular Polymer Systems ledby professor Petr Štěpánek, from 2009. She obtained her PhD in molecularphysics and thermophysics in 2006 at the Heat Physics Department ofUzbekistan AS. She is skilled in a number of scattering techniques, especiallydynamic and static light scattering, small angle neutron and x-ray scattering aswell in ultrasonic spectroscopy and calorimetric techniques. At present timeshe participates at investigation of “smart” self-assembling water-soluble poly-mers, such as thermo- and pH-responsive polymers and polymers for biomed-ical purposes.

Vladimir Kral is Professor of analytical chemistry at the Faculty of ChemicalEngineering, University of Chemistry and Technology in Prague (since 1995)and 1st Medical School, Charles University in Prague. He spent stays atMedical School, University of Texas at San Antonio, SA, USA;Pharmacylic, California, USA; Max-Planck Institute for Medicinal Research,Heidelberg, Germany; University MC of Paris 6, France; Technical UniversityMunchen, Germany and University of Kyoto, Japan. Major field of activities:Drug design, supramolecular chemistry, drug delivery systems, photodynamictherapy.

Martin Hrubywas born in Prague, Czech Republic, in 1978. He obtained hismaster degree with honors in the field of macroporous chelating polymers in2002 at the Faculty of Natural Sciences, Charles University in Prague. Hefinished his Ph.D. in 2006 at the Institute of Chemical Technology in Praguein the field of polymeric drug delivery systems. He also stayed at theUniversity of California at Berkeley (1999) and the Institute of NuclearChemistry and Technology, Warsaw (2007). He currently works as a SeniorScientist in the Department of Supramolecular Polymer Systems led byProfessor Petr Štěpánek at the Institute of Macromolecular Chemistry, ASCR, in the field of biocompatible polymers as drug and radionuclide carriers.Major field of activities: Preparation, characterisation and biological testing ofpolymeric carriers for diagnostic and therapeutic radiopharmaceuticals with aspecial focus on systems sensitive to changes of environment.

Colloid Polym Sci

Petr Stepanek graduated at the Charles University in Prague in chemical physicsand then obtained a doctoral degree in experimental physics of polymers. Hispostdoctoral research 1981-1982 was carried out in France at the Commissariat àl’Energie Atomique in Saclay where he used dynamic light scattering and small-angle neutron scattering to study fundamental properties of polymer solutions, thetheory of which was being developed at that time by prof. P. G. de Gennes. Hespent his sabbatical in 1989-1990 as a visiting scientist at the University ofMinnesota in Minneapolis where he conducted research on critical phenomena inpolymer blends and dynamics of block copolymers. After returning to the Instituteof Macromolecular Chemistry of the Academy of Sciences of the Czech Republiche took in 1997 the position of Head of Department of Supramolecular PolymerSystems where he developed experimental studies of self-organization in polymersystems. Since 2007 he acts as Deputy Director of the Institute.

His current research interests are focused to self-association phenomena inmacromolecular materials and the characterization of functional polymeric mate-rials for biomedical applications. His research team performs synthesis and char-acterization of tailored self-associated polymer systems for diagnostic and thera-peutic applications (including fluorescently and radioactively labeled polymers),biodegradable polymers for drug delivery, miktoarm and block-gradient polymers

forming complex structures. Special attention is paid to polymeric systems respon-sive to external stimuli, in particular to changes in pH and temperature or chemicalenvironment. His main experimental techniques used for investigation of polymersystems are scattering of light, X-rays and of neutron and synchrotron radiation.

Miroslav Slouf has been working at the Institute of MacromolecularChemistry of the Academy of Sciences of the Czech Republic since 1998.At 2002 he became a head of the electron microscopy group, which was latertransformed to the Department of morphology and rheology of polymer ma-terials. He obtained his PhD at the Charles University in Prague (2001; disser-tation focused on X-ray diffraction and charge densities of single crystals) andhis Assoc. Prof. title at the Brno University of Technology (2013; habilitationdealing with the development of highly-crosslinked UHMWPE for total jointreplacements with longer lifetime). At present, his main research interests arebulk polymers materials for medical applications (such as total joint replace-ments and local delivery of antibiotics) with the focus on their morphology,electron microscopy and micromechanical properties.

Colloid Polym Sci